Methods and compositions for preparing nucleic acids that preserve spatial-proximal contiguity information

ABSTRACT

Provided herein are methods and compositions for preparing nucleic acids in samples that preserve spatial-proximal contiguity information. Samples include, but are not limited to, formalin-fixed paraffin-embedded (FFPE) samples, deeply formalin-fixed samples and samples that comprise protein:cfDNA complexes.

RELATED PATENT APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.16/689,002, filed Nov. 19, 2019, entitled METHODS AND COMPOSITIONS FORPREPARING NUCLEIC ACIDS THAT PRESERVE SPATIAL-PROXIMAL CONTIGUITYINFORMATION, naming Anthony Schmitt, Catherine Tan, Derek Reid, Chris DeLa Torre and Siddarth Selvaraj as inventors and assigned attorney docketno. AMG-1003-UT, which claims the benefit of U.S. Provisional PatentApplication No. 62/785,643, filed Dec. 27, 2018, entitled METHODS ANDCOMPOSITIONS FOR PREPARING NUCLEIC ACIDS THAT PRESERVE SPATIAL-PROXIMALCONTIGUITY INFORMATION, naming Anthony Schmitt, Catherine Tan, DerekReid, Chris De La Torre and Siddarth Selvaraj as inventors and assignedattorney docket no. AMG-1003-PV2, and also claims the benefit of U.S.Provisional Patent Application No. 62/770,135, filed Nov. 20, 2018,entitled METHODS FOR PREPARING NUCLEIC ACIDS THAT PRESERVESPATIAL-PROXIMAL CONTIGUITY INFORMATION, naming Anthony Schmitt,Catherine Tan, Derek Reid, Chris De La Torre and Siddarth Selvaraj asinventors and assigned attorney docket no. AMG-1003-PV. This applicationis also related to U.S. Provisional Patent Application No. 62/589,505,filed Nov. 21, 2017, entitled PRESERVING SPATIAL-PROXIMAL CONTIGUITY ANDMOLECULAR CONTIGUITY IN NUCLEIC ACID TEMPLATES, naming SiddarthSelvaraj, Anthony Schmitt and Bret Reid as inventors and assignedattorney docket no. AMG-1002-PV. This patent application is also relatedto PCT Application No. PCT/US18/62005, filed Nov. 20, 2018, entitledPRESERVING SPATIAL-PROXIMAL CONTIGUITY AND MOLECULAR CONTIGUITY INNUCLEIC ACID TEMPLATES naming Siddarth Selvaraj, Anthony Schmitt andBret Reid as inventors and assigned attorney docket no. AMG-1002-PC. Theentire content of the foregoing patent applications is incorporatedherein by reference, including all text, tables and drawings.

FIELD

This technology relates to sequencing nucleic acids.

BACKGROUND

Next-generation sequencing (NGS) has emerged as the predominant set ofmethods for determining nucleic acid sequence for a plethora of researchand clinical applications. The typical NGS workflow is as follows: thenative genomic DNA, often organized as chromosome(s), is isolated fromthe nucleic acid source leading to its fragmentation, to produce nucleicacid templates which are subsequently read by a sequencing instrument togenerate sequence data.

SUMMARY

The technology pertains to methods for preparing nucleic acids in such away that preserves DNA spatial-proximal contiguity sequence informationenabling the detection of spatially proximal nucleic acids (e.g. HiC),serving applications that benefit from long-range sequence contiguityinformation (e.g. haplotype phasing, genomic rearrangement detection andother applications that are enabled by long-range sequence contiguityinformation).

Preserving spatial-proximal contiguity information during thepreparation of DNA from a sample of interest allows preservingcontiguity in sequencing data obtained therefrom. Contiguity-preservedsequencing data enables comprehensive determination of nucleic acidsequence, as manifested in the contiguity-preserved nucleic acidtemplate, by enabling identification of genomic variants, determinationof contiguity information to inform genome assemblies de novo,deconvolution of haplotype phase information, genomic rearrangementdetection, which together are fundamental to understand the role ofgenetics in living systems.

Formalin-fixed paraffin-embedded samples are typically not successfullyprepared using the initial steps of standard protocols designed forcells, i.e., cells that are not formalin-fixed paraffin-embedded (seeExample 18).

Provided in certain aspects is a method for preparing nucleic acids froma formalin-fixed paraffin-embedded (FFPE) sample, that preservesspatial-proximal contiguity information, comprising: a) providing aformalin-fixed paraffin-embedded sample; b) de-waxing the sample toproduce a dewaxed sample; c) rehydrating the dewaxed sample, therebygenerating a dewaxed/rehydrated sample; d) contacting thedewaxed/rehydrated sample with lysis buffer, thereby generating a lysedsample; e) contacting the lysed sample with denaturing detergent at atemperature greater than 65° C., thereby generating a solubilized anddecompacted sample; and f) contacting the solubilized and decompactedsample with one or more reagents that preserve spatial-proximalcontiguity information in the nucleic acids of the solubilized anddecompacted sample.

Also provided in certain aspects is a method wherein thedewaxed/rehydrated sample is contacted with an extracellular matrixprotease prior to contact with lysis buffer.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a formalin-fixed paraffin-embedded (FFPE) sample, that preservesspatial-proximal contiguity information, comprising: a) providing aformalin-fixed paraffin-embedded sample; b) de-waxing the sample toproduce a dewaxed sample; c) rehydrating the dewaxed sample, therebygenerating a dewaxed/rehydrated sample; d) contacting thedewaxed/rehydrated sample with denaturing detergent at a temperaturegreater than 65° C., thereby generating a solubilized and decompactedsample; and e) contacting the solubilized and decompacted sample withone or more reagents that preserve spatial-proximal contiguityinformation in the nucleic acids of the solubilized and decompactedsample.

Also provided in certain aspects is a method wherein thedewaxed/rehydrated sample is contacted with an extracellular matrixprotease prior to contact with denaturing detergent.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a formalin-fixed paraffin-embedded (FFPE) sample, that preservesspatial-proximal contiguity information comprising: a) providing aformalin-fixed paraffin-embedded sample, that has not beendewaxed/rehydrated; b) contacting the formalin-fixed paraffin-embeddedsample with lysis buffer, thereby generating a lysed sample; c)contacting the lysed sample with denaturing detergent at a temperaturegreater than 65° C., thereby generating a solubilized and decompactedsample; and d) contacting the solubilized and decompacted sample withone or more reagents that preserve spatial-proximal contiguityinformation in the nucleic acids of the solubilized and decompactedsample.

Also provided in certain aspects is a method wherein the sample iscontacted with an extracellular matrix protease prior to contact withlysis buffer.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a formalin-fixed paraffin-embedded (FFPE) sample, that preservesspatial-proximal contiguity information comprising: a) providing aformalin-fixed paraffin-embedded sample, that has not beendewaxed/rehydrated; b) contacting the formalin-fixed paraffin-embeddedsample with denaturing detergent at a temperature greater than 65° C.,thereby generating a solubilized and decompacted sample; and c)contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

Also provided in certain aspects is a method wherein the sample iscontacted with an extracellular matrix protease prior to contact withdenaturing detergent.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a deeply formalin-fixed sample, that preserves spatial-proximalcontiguity information comprising: a) providing a deeply formalin-fixedsample; b) contacting the deeply formalin-fixed sample with lysisbuffer, thereby generated a lysed sample; c) contacting the lysed samplewith denaturing detergent at a temperature greater than 65° C., therebygenerating a solubilized and decompacted sample; and d) contacting thesolubilized and decompacted sample with one or more reagents thatpreserve spatial-proximal contiguity information in the nucleic acids ofthe solubilized and decompacted sample.

Also provided in certain aspects is a method wherein the deeplyformalin-fixed sample is contacted with an extracellular matrix proteaseprior to contact with lysis buffer.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a deeply formalin-fixed sample, that preserves spatial-proximalcontiguity information comprising: a) providing a deeply formalin-fixedsample; b) contacting the deeply formalin-fixed sample with denaturingdetergent at a temperature greater than 65° C., thereby generating asolubilized and decompacted sample; and c) contacting the solubilizedand decompacted sample with one or more reagents that preservespatial-proximal contiguity information in the nucleic acids of thesolubilized and decompacted sample.

Also provided in certain aspects is a method wherein the deeplyformalin-fixed sample is contacted with an extracellular matrix proteaseprior to contact with denaturing detergent.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a formalin-fixed paraffin-embedded (FFPE) sample, that preservesspatial-proximal contiguity information, comprising: a) providing aformalin-fixed paraffin-embedded sample; b) dewaxing the sample toproduce a dewaxed sample; c) rehydrating the dewaxed sample, therebygenerating a dewaxed/rehydrated sample; d) contacting thedewaxed/rehydrated sample with an extracellular matrix protease; therebygenerating a dissociated sample; e) contacting the dissociated samplewith lysis buffer, thereby generating a lysed sample; f) contacting thelysed sample with sodium dodecyl sulfate (SDS) at a temperature of 74°C. for 40 minutes, thereby generating a solubilized and decompactedsample; and g) contacting the solubilized and decompacted sample withone or more reagents that generate proximity ligated nucleic acidmolecules in situ, thereby preserving spatial-proximal contiguityinformation in the nucleic acids of the solubilized and decompactedsample.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a formalin-fixed paraffin-embedded (FFPE) sample of cells, thatpreserves spatial-proximal contiguity information comprising: a)providing a formalin-fixed paraffin-embedded sample of cells; b)de-waxing the sample to produce a dewaxed sample; c) rehydrating thedewaxed sample, thereby generating a dewaxed/rehydrated sample; d)contacting the dewaxed/rehydrated sample with lysis buffer, therebygenerating a lysed sample; e) contacting the lysed sample withdenaturing detergent at a temperature of 62° C. for greater than 10minutes, thereby generating a solubilized and decompacted sample; and f)contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a formalin-fixed paraffin-embedded (FFPE) sample of cells, thatpreserves spatial-proximal contiguity information comprising: a)providing a formalin-fixed paraffin-embedded sample of cells; b)de-waxing the sample to produce a dewaxed sample; c) rehydrating thedewaxed sample, thereby generating a dewaxed/rehydrated sample; d)contacting the dewaxed/rehydrated sample; with denaturing detergent at atemperature of 62° C. for greater than 10 minutes, thereby generating asolubilized and decompacted sample; and e) contacting the solubilizedand decompacted sample with one or more reagents that preservespatial-proximal contiguity information in the nucleic acids of thesolubilized and decompacted sample.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a deeply formalin-fixed sample of cells, that preservesspatial-proximal contiguity information comprising: a) providing adeeply formalin-fixed sample of cells; b) contacting the deeplyformalin-fixed sample with lysis buffer, thereby generated a lysedsample; c) contacting the lysed sample with denaturing detergent at atemperature of 62° C. for greater than 10 minutes, thereby generating asolubilized and decompacted sample; and d) contacting the solubilizedand decompacted sample with one or more reagents that preservespatial-proximal contiguity information in the nucleic acids of thesolubilized and decompacted sample.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a deeply formalin-fixed sample of cells, that preservesspatial-proximal contiguity information comprising: a) providing adeeply formalin-fixed sample of cells; b) contacting the deeplyformalin-fixed sample with denaturing detergent at a temperature of 62°C. for greater than 10 minutes, thereby generating a solubilized anddecompacted sample; and c) contacting the solubilized and decompactedsample with one or more reagents that preserve spatial-proximalcontiguity information in the nucleic acids of the solubilized anddecompacted sample.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a formalin-fixed paraffin-embedded (FFPE) sample of cells, thatpreserves spatial-proximal contiguity information comprising: a)providing a formalin-fixed paraffin-embedded sample of cells, b)contacting the formalin-fixed paraffin-embedded sample with lysisbuffer, thereby generating a lysed sample; c) contacting the lysedsample with denaturing detergent at a temperature of 62° C. for greaterthan 10 minutes, thereby generating a solubilized and decompactedsample; and d) contacting the solubilized and decompacted sample withone or more reagents that preserve spatial-proximal contiguityinformation in the nucleic acids of the solubilized and decompactedsample.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a formalin-fixed paraffin-embedded (FFPE) sample of cells, thatpreserves spatial-proximal contiguity information comprising: a)providing a formalin-fixed paraffin-embedded sample of cells, b)contacting the formalin-fixed paraffin-embedded sample with denaturingdetergent at a temperature of 62° C. for greater than 10 minutes,thereby generating a solubilized and decompacted sample; and c)contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a sample comprising protein:cfDNA complexes, that preservesspatial-proximal contiguity information, comprising: a) providing asample comprising protein:cfDNA complexes; b) crosslinking theprotein:cfDNA complexes to neighboring protein:cfDNA complexes; and c)contacting the crosslinked protein:cfDNA complexes with one or morereagents that preserve spatial-proximal contiguity information in thecell free DNA of the sample.

Also provided in certain aspects is a method for preparing nucleic acidsfrom a sample comprising protein:cfDNA complexes, that preservesspatial-proximal contiguity information, comprising: a) providing asample comprising protein:cfDNA complexes; b) contacting the sample witha solid phase, thereby generating protein:cfDNA complexes associatedwith a solid phase; c) crosslinking the protein:cfDNA complexes toneighboring protein:cfDNA complexes or to the solid phase; and d)contacting the crosslinked protein:cfDNA complexes with one or morereagents that preserve spatial-proximal contiguity information in thecell free DNA of the sample.

Also provided in certain aspects are methods to preservespatial-proximal contiguity information comprising use of proximityligation, solid substrate-mediated proximity capture (SSPC),compartmentalization with or without a solid substrate or a Tn5tetramer.

Also provided in certain aspects are methods wherein nucleic acids withpreserved spatial-proximal contiguity information are sequenced toproduce sequence readouts. In certain aspects, the sequence readouts areutilized in applications that are based on the use of long-rangesequence contiguity information.

Also provided in certain aspects are methods wherein nucleic acids withpreserved spatial-proximal contiguity information are subjected tobisulfite treatment to generate bisulfite treated nucleic acids withpreserved spatial-proximal contiguity information.

Also provided in certain aspects are methods wherein the bisulfitetreated nucleic acids with preserved spatial-proximal contiguityinformation are sequenced to determine the methylation status of thenucleic acids with preserved spatial-proximal contiguity information.

Also provided in certain aspects are kits comprising reagents forperforming the methods described herein.

Also provided in certain aspects are methods for rapidly reversingcrosslinking in a sample cross-linked to preserve spatial-proximalcontiguity information.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate certain embodiments of the technology and arenot limiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIGS. 1A-D show the extension of chromatin solubilization anddecompaction reaction improves capture of spatial-proximal contiguitysignal from formalin-fixed paraffin-embedded (FFPE) samples. FIG. 1Ashows results of experiments with 10 um sections and reaction times of10 minutes and 40 minutes. FIG. 1B shows results of experiments with 10um sections and reaction times of 40 minutes, 60 minutes and 80 minutes.FIG. 1C shows results of experiments with 10 um sections and reactiontimes of 40 minutes, 80 minutes, 120 minutes and 180 minutes. FIG. 1Dshows results of experiments with 5 um sections and reaction times of 40minutes, 80 minutes, 120 minutes and 180 minutes.

FIGS. 2A and 2B show increasing the temperature of chromatinsolubilization and decompaction improves capture of spatial-proximalcontiguity signal from formalin-fixed paraffin-embedded (FFPE) samples.FIG. 2A shows the results of experiments with 5 um sections, at threetemperatures (50° C., 62° C. and 74° C.) and reaction times of 10minutes, 40 minutes and 80 minutes. FIG. 2B shows the results ofexperiments with 10 um sections, at a temperature of 74° C. and reactiontimes of 10 minutes, 40 minutes and 80 minutes.

FIG. 3 shows chromatin solubilization and decompaction at 74° C. for 40minutes optimally captures spatial-proximal contiguity signal in aclinical human FFPE tumor sample.

FIG. 4 shows cellular lysis is not required to optimally capturespatial-proximal contiguity signal in FFPE samples.

FIGS. 5A and 5B show the effect of time and temperature of the SDSreaction on the need for de-waxing and rehydration to optimally capturespatial-proximal contiguity from FFPE samples. FIG. 5A shows de-waxingand rehydration is required to capture spatial-proximal contiguitysignal from FFPE samples when the SDS reaction is at 62° C. FIG. 5Bshows de-waxing and rehydration is not required to capturespatial-proximal contiguity signal from FFPE samples when the SDSreaction is at 74° C. and longer duration.

FIG. 6 shows enzymatic dissociation of extracellular matrix proteinsimproves ease of sample use without compromising optimal capture ofspatial-proximal contiguity signal from FFPE samples.

FIG. 7 illustrates protein:cfDNA complexes after binding to acarboxylated solid phase element and protein:cfDNA crosslinking.

FIG. 8 illustrates protein:cfDNA complexes after crosslinking to a solidphase element coated with a nucleic acid crosslinking reagent.

FIG. 9 illustrates capturing spatial-proximal contiguity informationfrom protein:cfDNA complexes via proximity ligation.

FIG. 10 illustrates using Tn5 tetramer to capture to capturespatial-proximal contiguity information from crosslinked protein:cfDNAcomplexes.

FIG. 11 illustrates using Tn5 tetramer to capture spatial-proximalcontiguity information for cell free nucleic acids crosslinked to solidphase element (e.g., via psoralen).

FIG. 12 illustrates capturing spatial-proximal contiguity informationfrom protein:cfDNA complexes via compartmentalization and tagging withcompartment-specific molecular barcodes.

FIG. 13 illustrates capturing spatial-proximal contiguity informationfrom protein:cfDNA complexes via compartmentalization with solid phaseelement and tagging with compartment-specific molecular barcodes.

FIG. 14 illustrates capturing spatial-proximal contiguity informationfrom protein:cfDNA complexes via virtual compartmentalization usingbead-linked transposome carrying virtual compartment-specific molecularbarcodes.

FIG. 15 illustrates capturing spatial-proximal contiguity informationfrom FFPE samples via proximity ligation.

FIG. 16 illustrates capturing spatial-proximal contiguity informationfrom FFPE samples via SSPC methods.

FIG. 17 illustrates capturing spatial-proximal contiguity informationfrom FFPE samples via compartmentalization and tagging withcompartment-specific molecular barcodes.

FIG. 18 shows failure of chromatin digestion in FFPE cells.

FIG. 19 shows automated HiC on FFPE tissues.

FIG. 20 shows rapid reverse crosslinking in FFPE tissues.

FIG. 21 shows highly sensitive discovery of a known translocation as afunction of mutant allele frequency and sequencing depth usinggenome-wide HiC data from FFPE cells.

FIG. 22 shows highly sensitive discovery of a known translocationenabled by targeted HiC data from FFPE cells.

FIGS. 23A-D show discovery and validation of translocations in an FFPEGIST tumor. FIG. 23A show shallow sequencing analysis (0.75×). FIG. 23Bshows sequence analysis at 10×. FIG. 23C shows amplification across thetranslocation breakpoint. FIG. 23D shows PCR results.

FIGS. 24A-C show discovery of translocations in an FFPE pediatricependymoma tumor. FIG. 24A shows HiC data from PFE cell lines. FIG. 24Bshows shallow sequencing analysis (0.25×). FIG. 24C shows FFPE-HiCanalyses of a PFE tumor.

FIGS. 25A-D show discovery of translocations in FFPE tumors acrossarchival periods. FIG. 25A shows shallow sequencing analysis (0.05×).FIG. 25B shows intra-chromosome translocations. FIG. 25C showsinter-chromosome translocation between chr3; chr18. FIG. 25D showsinter-chromosome translocation between chr3; chr 7 translocation

FIG. 26 shows high quality FFPE-HiC from low input FFPE tissue.

DETAILED DESCRIPTION

Provided herein are methods for preparing nucleic acids from particulartypes of samples that preserves spatial-proximal contiguity informationin the sequence of the nucleic acids. Nucleic acid molecules thatpreserve spatial-proximal contiguity information can fragmented andsequenced using short-read sequencing methods (e.g. Illumina, nucleicacid fragments of lengths approximately 500 bp) or intact molecules thatpreserve spatial-proximal contiguity information can be sequenced usinglong-read sequencing (e.g. Pacific Bioscience (now Illumina), OxfordNanopore, or others, nucleic acid fragments of lengths approximately 30Kbp or greater).

In certain embodiments, a sample can be a fixed sample that is embeddedin a material such as paraffin (wax). In some embodiments, a sample canbe a formalin fixed sample. In certain embodiments, a sample isformalin-fixed paraffin-embedded sample. In some embodiments, aformalin-fixed paraffin-embedded sample can be a tissue sample or a cellculture sample. In some embodiments, a tissue sample has been excisedfrom a patient and can be diseased or damaged. In some embodiments, atissue sample is not known to be diseased or damaged. In certainembodiments, a formalin-fixed paraffin-embedded sample can be aformalin-fixed paraffin-embedded section, block, scroll or slide. Incertain embodiments, a sample can be a deeply formalin-fixed sample, asdescribed below.

In certain embodiments, a formalin-fixed paraffin-embedded sample isprovided on a solid surface and a method of preparing nucleic acid thatpreserves spatial-proximal contiguity information is performed on thesolid surface. In some embodiments, a solid surface is a pathologyslide. In some embodiments, additional downstream reactions are alsoperformed on the solid surface. Those of skill in the art are familiarwith methods that can be substituted for steps requiring centrifugationand that achieve a comparable result, but are performed on a solidsurface.

Low Input DNA

Often it is difficult to obtain even small amounts of DNA from FFPEsamples (input DNA) that is of a quality that allows forspatial-proximity analysis, such as HiC analysis. In certainembodiments, utilizing the optimized protocols described herein, DNA isextracted from an FFPE sample or a deeply formalin-fixed sample, even ina low amount, that produces sufficient long-range cis readouts forrobust spatial-proximity analysis and use in other applications (seeFIG. 26). In certain embodiments, the input DNA extracted from a sampleis less than 200 ng. In certain embodiments, the amount of input DNAextracted from a sample is less than 160 ng, less than 120 ng, less than80 ng, less than 40 ng, less than 20 ng, less than 10 ng, less than 5ng, less than 2 ng or less than 1 ng.

Extended Archival Periods

Prolonged FFPE archival periods or prolonged deeply formalin-fixedarchival periods are known to degrade DNA and make genomic analyses moretechnically challenging and therefore is a critical parameter toevaluate when developing a robust genomic analysis method for FFPEsamples and for deeply formalin-fixed samples. In certain aspects,utilizing the optimized protocols described herein, DNA is obtained fromlong archival periods that results in sufficient long-cis readouts forrobust spatial-proximity analysis and use in other applications (seeFIGS. 25A-D). In some embodiments, an extended archival period isgreater than about 1 year, greater than about 4 years, greater thanabout 10 years, greater than about 20 years, greater than about 30years, greater than about 40 years, greater than about 50 years, greaterthan about 60 years or greater than about 70 years, or sometimes about1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79 or 80 years. In certain embodiments, the sample hasan archival period of about 4 years to about 20 years. In certainembodiments, the sample has an archival period of about 20 years toabout 70 years. In some embodiments, the archival period can be lessthan about 4 years, less than about 3 years, less than about 2 years orless than about 1 year, if the sample is of low quality, i.e., the DNAof the sample is degraded to an extent often observed for samples withlonger archival periods.

Preparation of Formalin-Fixed Paraffin Embedded (FFPE) Samples withDe-Waxing/Rehydrating

In certain embodiments, a formalin-fixed paraffin-embedded sample isdewaxed. De-waxing can be carried out by contacting the formalin-fixedparaffin-embedded sample with any known agent that dissolves wax. Insome embodiments, the agent is a solvent. In some embodiments, thesolvent is an organic solvent. In some embodiments, the solvent isxylene. In some embodiments, the solvent is toluene, benzene or anyother suitable solvent. In some embodiments, the agent is a non-toxicagent, including but not limited to mineral oil, or an agent withlow-toxicity, including but not limited to limonene.

In certain embodiments, a dewaxed formalin-fixed paraffin-embeddedsample is rehydrated. In certain embodiments, the dewaxed sample isrehydrated by contact with ethanol (or any agent that is useful forremoving a solvent from the sample) and re-suspended in water or asuitable buffer.

In some embodiments, a dewaxed/rehydrated formalin-fixedparaffin-embedded sample is contacted with a lysis buffer prior tocontact with a denaturing detergent. As used herein, the term “lysisbuffer” refers to a buffered solution able to lyse cell membranes. Lysisbuffers typically comprise salts, a protease inhibitor and a non-ionic,non-denaturing detergent and are known by those who practice the art. Incertain embodiments the lysis buffer is hypotonic. In certainembodiments, the lysis buffer comprises a protease inhibitor. In certainembodiments the lysis buffer comprises a nonionic non-denaturingdetergent. In some embodiments, the nonionic non-denaturing detergent isIGEPAL or an equivalent detergent. In certain embodiments the lysisbuffer does not include a protease.

In certain embodiments, a dewaxed/rehydrated formalin-fixedparaffin-embedded sample is not contacted with a lysis buffer prior tocontact with a denaturing detergent. Without being held to a theory,whether lysis buffer is required may depend on the thickness of aformalin-fixed paraffin-embedded tissue section. In certain embodiments,the formalin-fixed paraffin-embedded tissue section is from about 5 toabout 10 um in thickness. In certain embodiments, the formalin-fixedparaffin-embedded tissue section is about 5 um or less in thickness. Atissue section with a thickness of about 5 um is less than the thicknessof a nucleus. Without being bound by theory, a tissue section with athickness of about 5 um slices through a nucleus and provides directaccess to the interior of the nucleus, thus cell lysis may not berequired. Lysis buffer may also not be required when contact withdenaturing detergent is at an elevated temperature (e.g., greater than65° C.) and/or for greater than 10 minutes.

In certain embodiments, there is no reagent utilized specifically todissociate or break apart the tissue of the sample. For example, thereis no enzymatic dissociation with a protease.

In certain embodiments, a dewaxed/rehydrated formalin-fixedparaffin-embedded sample is contacted with a protease. In certainembodiments, the dewaxed/rehydrated formalin-fixed paraffin-embeddedsample is contacted with the protease prior to contact with otherreagents (e.g., lysis buffer, denaturing detergent). In some embodimentsthe protease is an extracellular matrix (ECM) protease that dissociatesthe tissue of the sample. In some embodiments, the extracellular matrixprotease is collagenase and/or dispase. In certain embodiments thecollagenase is ColI ColIII or ColIV. In some embodiments the dispase isDispase I (Neutral Protease I). Without being bound to a theory, aprotease may dissociate extracellular matrix proteins in the sample,thus improving the ease of handling and transfer of the sample.

In some embodiments, a dewaxed/rehydrated formalin-fixedparaffin-embedded sample is contacted with a denaturing detergent. Asused herein, the term “denaturing detergent” refers to an anionic orcationic detergent. In certain embodiments the detergent can be sodiumdodecyl sulfate (SDS).

As used herein, the term “solubilized and decompacted sample” refers toa sample contacted with a denaturing detergent and having one or more ofthe following features: permeabilized nuclei, de-condensed chromatin(decompacted) and/or solubilized chromatin that has preservedspatial-proximal contiguity information.

In some embodiments, contact with a denaturing detergent is for greaterthan about 10 minutes, or about 15 to about 80 minutes, about 20 toabout 60 minutes, greater than 10 minutes to about 40 minutes, about 30to about 50 minutes, about 35 minutes to about 45 minutes or sometimesabout 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80minutes. In some embodiments, contact with a denaturing detergent is forabout 40 minutes. In some embodiments, contact with a denaturingdetergent is for 40 minutes.

In some embodiments, contact with a denaturing detergent is at atemperature of about 65° C. to about 90° C., about 70° C. to about 80°C. or sometimes about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90° C. In certainembodiments the temperature is 74° C. In some embodiments, contact witha detergent is at a temperature of greater than 65° C., at a temperatureof greater than 65° C. and less than 80° C., at a temperature of between70° C. and 80° C., or at a temperature of about 74° C. In someembodiments, the sample is a tissue sample.

In some embodiments, the sample comprises cells and contact with adenaturing detergent is at a temperature of about 62° C. for greaterthan 10 minutes. In some embodiments, the sample comprises cells andcontact with a denaturing detergent is at a temperature of about 62° C.for 40 minutes.

In certain embodiments, the sample comprises tissues and contact with adenaturing detergent is at a temperature of about 74° C. for 40 minutes.

Preparation of Formalin-Fixed Paraffin Embedded (FFPE) Samples WithoutDe-Waxing/Rehydrating

In certain embodiments, a formalin-fixed paraffin-embedded sample is notdewaxed and rehydrated. In some embodiments, a formalin-fixedparaffin-embedded sample that is not dewaxed and rehydrated is contactedwith lysis buffer and the lysed sample is contacted with denaturingdetergent. In some embodiments, a formalin-fixed paraffin-embeddedsample that is not dewaxed and rehydrated is not contacted with lysisbuffer and is directly contacted with denaturing detergent. In certainembodiments, a formalin-fixed paraffin-embedded sample that is notdewaxed and rehydrated is a tissue sample that is contacted withdenaturing detergent at a temperature that is greater than 65° C. Insome embodiments, the temperature is 74° C. In certain embodiments, aformalin-fixed paraffin-embedded tissue section that is not dewaxed andrehydrated is from about 5 to about 10 um in thickness. In certainembodiments, the formalin-fixed paraffin-embedded tissue section that isnot dewaxed and rehydrated is about 5 um or less in thickness.

In certain embodiments, a formalin-fixed paraffin-embedded sample thatis not dewaxed and rehydrated is provided on a solid surface. In someembodiments, the solid surface is a pathology slide.

Preparation of Deeply Formalin-Fixed Samples

In some embodiments, a sample is deeply fixed using formalin, but is notparaffin-embedded. For example, some surgical samples are fixed withformalin and preserved in a liquid solution. Accordingly, such a deeplyformalin-fixed sample does not require dewaxing and/or rehydration, butis otherwise typically processed the same as an FFPE sample.

In some embodiments, a deeply formalin-fixed sample is contacted withlysis buffer (as described herein) and the lysed sample is contactedwith denaturing detergent. In some embodiments, a deeply formalin-fixedsample is not contacted with lysis buffer (as described herein) and thesample is contacted with denaturing detergent. In some embodiments, adeeply formalin-fixed sample is contacted with a protease.

Preserving Spatial-Proximal Contiguity Information

In certain embodiments, formalin-fixed paraffin-embedded (FFPE) sampleswith and without de-waxing/rehydrating and deeply formalin-fixed samplesare processed using the described methods to generate a solubilized anddecompacted sample which is contacted with one or more reagents thatpreserve spatial-proximal contiguity information in the nucleic acids ofthe sample. In certain embodiments, the formalin-fixed paraffin-embedded(FFPE) samples and deeply formalin-fixed samples are samples of tissues.In certain embodiments, the formalin-fixed paraffin-embedded (FFPE)samples and deeply formalin-fixed samples are samples of cells.Regardless of the type of sample, the same reagents that preservespatial-proximal contiguity information are utilized.

As used herein, the term “reagents that preserve spatial-proximalcontiguity information” refers to reagents and their methods of use thatcapture and preserve the native spatial conformation exhibited bynucleic acids when associated with proteins as in chromatin and/or aspart of a nuclear matrix. Spatial-proximal contiguity information can bepreserved by proximity ligation, by solid substrate-mediated proximitycapture (SSPC), by compartmentalization with or without a solidsubstrate or by use of a Tn5 tetramer.

Proximity Ligation

In some embodiments, reagents that preserve spatial-proximal contiguityinformation are reagents that generate proximity ligated nucleic acidmolecules that are utilized in methodologies comprising proximityligation. A proximity ligation method is one in which natively occurringspatially proximal nucleic acid molecules are captured by ligation togenerate ligated products. In some embodiments, reagents that generateproximity ligated nucleic acid molecules can include a restrictionendonuclease, a DNA polymerase, a plurality of nucleotides comprising atleast one biotinylated nucleotide, and a ligase. In certain embodiments,there are two restriction endonucleases.

FIG. 15 shows capturing spatial-proximal contiguity information fromFFPE samples via PL (Proximity Ligation) methods. PL methods begin with(i) native spatially proximal nucleic acids (nSPNAs) within a nucleicacids source (e.g. FFPE sample), followed by (ii) digestion (e.g. viaRE) and ligation to generate ligation products (LPs). Note that for FFPEsamples that are already highly fragmented, digestion of the DNA may notbe necessary. Broadly, PL methods are classified as 3C-based andHiC-based, although there are many specific variations of PL. In 3C(iii), the plurality of LPs are fragmented, prepared as short nucleicacid templates and ready for sequencing. In HiC (iv), the digestednucleic acid ends are marked (e.g. biotinylated) and then ligated tocreate marked ligated products (MLPs, MLPs are a manifestation of LPs),bearing an affinity purification marker at the ligation junctions (LJs).After the plurality of MLPs are fragmented, affinity purification isused to enrich for fragments of MLPs comprising LJs and such fragmentsare prepared as nucleic acid templates and are ready for sequencing—i.e.the fragmented nucleic acids from the MLPs that contain at least an LJare enriched and prepared as a template and sequenced in HiC, to depleteuMLPs (unligated MLPs that do not usually manifest LJs).

Methods of carrying out proximity ligation are known in the art. Forexample, in the HiC method steps typically include: (1) digestion ofchromatin of the solubilized and decompacted sample with a restrictionenzyme (or fragmentation); (2) labelling the digested ends by filling inthe 5′-overhangs with biotinylated nucleotides; and (3) ligating thespatially proximal digested ends, thus preserving spatial-proximalcontiguity information. Once spatial-proximal contiguity information ispreserved, further steps in the HiC method include: purifying andenriching the biotin-labelled ligation junction fragments, preparing alibrary from the enriched fragments and sequencing the library. (seeLieberman-Aiden et al. US2017/0362649, Lieberman-Aiden et al. Science326, 289-293 (2009), Dekker et al. (U.S. Pat. No. 9,434,985)). (see FIG.15) Another example of a proximity ligation method, often includessteps: (1) digestion of chromatin of the solubilized and decompactedsample with a restriction enzyme (or fragmentation); (2) blunting thedigested or fragmented ends or omission of the blunting procedure; and(3) ligating the spatially proximal ends, thus preservingspatial-proximal contiguity information. Once spatial-proximalcontiguity information is preserved, further steps can include: usingsize selection to purify and enrich ligated fragments, which representligation junction fragments, preparing a library from the enrichedfragments and sequencing the library.

In some embodiments, the proximity ligated nucleic acid molecules aregenerated in situ. As used herein the term “in situ” refers to within anucleus (see U.S. Application US2017/0362649).

Proximity ligation methods include, but are not limited to 3C (Dekker etal. Science 295, 1306-1311 (2002), 4C (Simonis et al. Nature Genetics38, 1348-1354 (2006), De Laat et al. (U.S. Pat. No. 8,642,295)) 5C(Dostie et al. Genome Research 16, 1299-1309 (2006), Dekker et al. (U.S.Pat. No. 9,273,309), HiC (Lieberman-Aiden et al. US2017/0362649,Lieberman-Aiden et al. Science 326, 289-293 (2009), Dekker et al. (U.S.Pat. No. 9,434,985), TCC (Kalhor et al. Nature Biotechnology 30, 90-98(2012), Chen et al. (US20110287947) 4C-seq (Van de Werken et al. NatMethods. (2012)), ChIA-PET (Ruan et al U.S. Pat. No. 8,071,296), HiChIP(Mumbach et al. Nat Methods. (2016) PLAC-seq (Fang et al. Cell Research(2016)), Capture-C (Hughes et al. Nature Genetics (2014), Capture-HiC(Jager et al. Nature Communications, 2015), or other methods orcombination of methods.

Regardless of the specific PL method, all PL methods capturespatial-proximal contiguity information in the form of ligationproducts, whereby a ligation junction is formed between two nativelyspatially proximal nucleic acids. Once the LPs are formed, thespatial-proximal contiguity information is detected using nextgeneration sequencing, whereby one or more ligation junctions (eitherfrom an entire LP or fragment of an LP) are sequenced (as describedherein). With these sequence information, one is informed that thenucleic acid molecules from a given ligation product (or ligationjunction) are natively spatially proximal nucleic acids.

Solid Substrate-Mediated Proximity Capture (SSPC)

In some embodiments, reagents that preserve spatial-proximal contiguityinformation are reagents that comprise solid substrates that formcomplexes with the nucleic acid of the solubilized and decompactedsample and are utilized in methodologies comprising solidsubstrate-mediated proximity capture (SSPC). SSPC methods compriseintroducing an exogenous solid substrate functionalized with surfacemolecule(s) that captures natively occurring spatially proximal nucleicacid molecules by binding to them. In some embodiments of solidsubstrate-mediated proximity capture (SSPC) a sample is contacted withsolid substrates that form complexes with the nucleic acid of the sampleprior to the sample being solubilized and decompacted.

FIG. 16 illustrates of capturing spatial-proximal contiguity informationfrom FFPE samples via SSPC methods. SSPC methods comprise introducing anexogenous solid substrate functionalized with surface molecule(s) thatcaptures nSPNAs by binding them. In all cases, the solid substrate isintroduced into a source of nucleic acids (e.g. FFPE sample), and in (i)the solid substrate is functionalized with a nucleic acid crosslinkingagent such that the surface of the solid substrate becomes chemicallybound to the nSPNAs for which it physically contacts. In (ii) thenucleic acids of the nucleic acid source are first labeled with anaffinity purification marker and then a solid substrate functionalizedwith an affinity purification molecule is introduced such that thesurface of the solid substrate becomes chemically bound to the labelednSPNAs for which it physically contacts. In (iii) the solid substrate isfunctionalized with transposase bearing barcoded oligonucleotides, suchthat each solid substrate has its own set of uniquely barcodedoligonucleotides, and such that when the surface of the solid substratecomes in physical contact with nSPNAs, the barcoded oligonucleotides areintegrated into nSPNAs.

As used herein, the term “solid substrate” refers to, for example, beadsor other small solid phase particles or surfaces.

In certain embodiments, the solid substrates are solid substratesfunctionalized (e.g., coated) with a nucleic acid crosslinking agent(see FIG. 16, left panel). The surface of the solid substrate becomeschemically bound to the natively occurring spatially proximal nucleicacid molecules for which it physically contacts. Crosslinking agents areknown in the art. In some embodiments the crosslinking reagent ispsoralen. Spatial-proximal contiguity information is preserved bycompartmentalizing (see below) and tagging molecules bound to a commonsolid phase substrate with a unique compartment specific molecularbarcode (see below).

In some embodiments, nucleic acids are first labeled with an affinitypurification marker (e.g. biotin), and a solid substrate isfunctionalized with an affinity purification molecule capable of bindingthe affinity purification marker (e.g. streptavidin) (see FIG. 16,middle panel) Similar to the aforementioned crosslinking-based SSPCmethod, spatial-proximal contiguity information is preserved bycompartmentalizing and tagging molecules bound to a common solid phasesubstrate with a unique compartment-specific molecular barcode (seebelow).

In certain embodiments, the solid substrate is functionalized withtransposases comprising barcoded oligonucleotides (e.g., Tn5). Eachsolid substrate has its own set of uniquely barcoded oligonucleotides,such that when the surface of the solid substrate comes in physicalcontact with nucleic acid molecules in the solubilized and decompactedsample the barcoded oligonucleotides are integrated into nucleic acidmolecules, thus preserving spatial-proximal contiguity information (seeFIG. 16, right panel). This is an example of “virtual”compartmentalization, as the uniquely barcoded transposases affixed tothe solid substrate can be thought to represent its own “virtual”compartment, within which are a collection of uniquely barcoded nucleicacid molecules that represent spatial-proximal contiguity information(e.g. as in CPT-seqV2 (Zhang et al. Nature biotechnology 35, 852-857(2017)). After tagging is completed, the transposome protein istypically denatured and thus would release the barcoded spatiallyproximal nucleic acid molecules from the bead-linked transposome. Insome embodiments, denaturing is by contact with a detergent (e.g., 0.2%SDS for 15-30 minutes at about 55° C.) or contact with a chaotropic salt(e.g., guanidine hydrochloride). In some embodiments after the barcodedspatially proximal nucleic acid molecules are released, the beads areremoved and the nucleic acid is purified (see below).

As used herein, the term “compartmentalizing” refers to the act ofpartitioning a plurality of nucleic acids which preservespatial-proximal contiguity information into a multitude of discretecompartments such that each compartment is allocated with a sub-haploidquantity of nucleic acids. In cases of “physical” compartmentalization,a plurality of nucleic acids can be partitioned into discrete physicalspaces (i.e. compartments) that are barred from intermixing with othercompartments. Such a physical compartment might be the well of amicrotiter plate (e.g. as in CPT-Seq (Adey et al. Genome Research 24,2041-2049 (2014) and Amini et al. Nature Genetics 46 1343-1349 (2014))),a microfluidic droplet (e.g. as in 10× Genomics (Zheng et al. NatureBiotechnology 34, 303-311 (2016))) or other compartmentalizationreagents. Compartmentalization can be carried out with nativelyoccurring spatially proximal nucleic acid molecules crosslinked to asolid substrate or bound to a solid substrate via the interaction of anaffinity purification molecule and an affinity purification marker.Spatially proximal nucleic acid molecules crosslinked or bound to acommon solid phase substrate can be captured in a droplet (thuscompartmentalized). In some embodiments, compartmentalization does notrequire the spatially proximal nucleic acid molecules be associated witha solid substrate.

As used herein, the term “tagging” refers to physically integratingunique molecular identifiers (i.e. molecular barcodes, defined below) aspart of (or in amplicons of) the nucleic acids which preservespatial-proximal contiguity information . As described herein, molecularbarcodes can be integrated into nucleic acids of interest usingtransposases to integrate a uniquely barcoded oligonucleotide into thenucleic acid molecule or, via techniques such as primer extensionpolymerization (PEP), where a polymerase and a primer comprising amolecular barcode anneals to and extends along the nucleic acidmolecule, thereby creating amplicons of the nucleic acid molecule thatare contiguous with the barcoded primer nucleic acids. Also described isan alternate form of tagging involving the ligation of anoligonucleotide comprising a molecular barcode to a terminal end(s) of anucleic acid molecule. Tagging of the spatially proximal nucleic acidmolecules can be carried out with the spatially proximal nucleic acidmolecules associated with a solid substrate.

As used herein, the term “molecular barcode” refers to a uniquelyidentifiable nucleic acid sequence that uniquely informs the context forwhich the molecular barcode was introduced. For example, when amolecular barcode is integrated into a nucleic acid molecule andsubsequently sequenced, the molecular barcode manifested in thesequencing readout informs about the compartment or virtual compartmentwith which the nucleic acid molecule was associated and thus containsspatial-proximal contiguity information.

Typically, after compartmentalization and tagging, the compartments(e.g., droplets) would be merged together (for example, droplets wouldburst and combine into a single intermixing sample) and any protein maybe denatured (e.g. ProK treatment) to release the spatially proximalnucleic acid molecules from a solid substrate. The solid substrate(e.g., beads) could be removed magnetically, if the beads are magnetic,or pelleted, if the beads are not magnetic. The spatially proximalnucleic acid molecules would be purified using standard methods (e.g.ethanol precipitation, SPRI beads, Qiagen columns, etc.).

Regardless of the specific SSPC method, all SSPC methods capturespatial-proximal contiguity information in compartment-specific orvirtual compartment-specific molecular barcode. Once the SSPC productsare formed, the spatial-proximal contiguity information is detectedusing DNA sequencing, whereby two or more common molecular barcodesequences and the contiguous adjacent nucleic acids are sequenced (asdescribed herein). With these sequence information, one is informed thatthe nucleic acid molecules adjoined to a common molecular barcode arenatively spatially proximal nucleic acids.

Compartmentalization Without a Solid Substrate

In some embodiments, reagents that preserve spatial-proximal contiguityinformation are reagents that compartmentalize and tag by attachingcompartment-specific molecular barcodes (as previously described), inthe absence of a solid substrate. In some embodiments,compartment-specific barcoded oligonucleotides are attached to nativelyspatially proximal nucleic acid molecules by ligation. In someembodiments, compartment-specific barcoded oligonucleotides are attachedto natively spatially proximal nucleic acid molecules by primerextension reactions.

FIG. 17 illustrates capturing spatial-proximal contiguity informationfrom FFPE samples via compartmentalization and tagging withcompartment-specific molecular barcodes. In one embodiment of themethod, the method begins with (i) native spatially proximal nucleicacids (nSPNAs), followed by (ii) compartmentalization of the crosslinkednSPNAs and introducing a compartment specific molecular barcode, such asligating a compartment-specific barcoded oligonucleotide. Finally (iii),barcoded template molecules are purified and prepared as nucleic acidtemplates and are ready for sequencing, whereby the molecular barcode isthe molecular identifier for which nSPNAs were spatially proximal.

Similar to the SSPC methods, spatial-proximal contiguity information isdetected using DNA sequencing, whereby two or more common molecularbarcode sequences and the contiguous adjacent nucleic acids aresequenced (as described herein). With these sequence information, one isinformed that the nucleic acid molecules adjoined to a common molecularbarcode are natively spatially proximal nucleic acids.

Tn5 tetramer

In certain embodiments, reagents that preserve spatial-proximalcontiguity information comprise a Tn5 tetramer (see Lai et al. NatureMethods. Vol. 15, September 2018, 741-747). Methods involving the use ofthe Tn5 tetramer (as described herein) capture spatial-proximalcontiguity information in the form of Tn5-mediated looping products,whereby a physical link, mediated by the Tn5 linker oligonucleotide, isformed between two natively spatially proximal nucleic acids. Once theTn5-mediated looping products are formed, the nucleic acid is releasedfrom Tn5 (see discussion above), the spatial-proximal contiguityinformation is detected using DNA sequencing, whereby the nucleic acidson both sides of a given Tn5 linker oligonucleotide are sequenced (asdescribed herein). With these sequence information, one is informed thatthe nucleic acid molecules from a given Tn5-mediated looping product arenatively spatially proximal nucleic acids. (see detailed descriptionbelow)

In certain embodiments, one or more steps of the methods describedherein is performed using automated equipment (see Example 19). In someembodiments, essentially all of the steps of the methods describedherein are performed using automated equipment.

Sequencing

In certain embodiments, nucleic acids with preserved spatial-proximalcontiguity information from formalin-fixed paraffin-embedded or deeplyformalin-fixed samples, as described herein, are sequenced to producesequence readouts. In some embodiments, sequencing is at a depth of 30×or less. In some embodiments, sequencing is at a depth of 15× or less,10× or less, 5× or less, 1× or less, 0.75× or less, 0.5× or less, 0.1×or less, 0.05× or less or 0.01× or less.

Quality of DNA Templates

One metric for the quality of the DNA templates from FFPE samples ordeeply formalin-fixed samples prepared utilizing the methods describedherein when further analyzed by methods that preserve spatial-proximalcontiguity information (e.g., proximity ligation methods, such as HiC)is the percent of long-cis readouts (long-range) readouts. In someembodiments, the criteria for assessing quality is that >40% of mappedde-duplicated reads represent long-range readouts of at least 15 kb. Insome embodiments, depending on the properties of the sample, such as theextent of the archival period, the criteria may be lower than greaterthan 40%, such about 20 to 25% long-range readouts (see FIG. 25A). Suchreadouts enable identification of translocations at low sequencing depth(se FIGS. 25B-D). In some embodiments, the criteria is greater than 35%,30%, 25%, 20% or 15% long-range readouts greater than 15 kb. In certainembodiments, the long range readout are defined as 100 kb-1 Mb, >20kb, >10 kb, >5 kb, or >1 kb in length and the respective percent oflong-range readouts will change depending on the definition oflong-range readouts. DNA templates prepared from FFPE samples or deeplyformalin-fixed samples utilizing the methods described herein result ina greater % of long-range readouts than when samples are preparedwithout utilizing the described methods for solubilizing anddecompacting (e.g., contact with a denaturing detergent at a temperaturegreater than 65° C., contact with a denaturing detergent at atemperature greater than 65° C. for greater than 10 minutes, contactwith a denaturing detergent at a temperature of 74° C. for 40 minutes,contact with a denaturing detergent at a temperature of about 62° C. forgreater than 10 minutes, contact with a denaturing detergent at atemperature of 62° C. for 40 minutes).

In certain embodiments, other metrics to measure quality of the DNAtemplates include measurements of frequency, sensitivity, specificity,false positive rate, etc., for example when identifying translocations.

Reversal of Crosslinking

In certain embodiments, crosslinking is reversed by incubation withprotease K (ProK) at a reduced temperature for a shortened period oftime. In some embodiments, the temperature is less than 68° C. and thetime is about 30 min. In some embodiments the temperature is about 55°C. In some embodiments the temperature is 55° C. (see Example 20).

In certain embodiments, crosslinking is reversed by incubating thesample at a temperature of about 95° C. for about 1hour in the absenceof proteinase K. In some embodiments, crosslinking is reversed byincubating the sample at a temperature of 95° C. for 1 hour in theabsence of proteinase K.

Preparation of Samples with Protein:cfDNA Complexes

In some embodiments, a sample comprises protein:cfDNA (protein:cell-freeDNA) complexes in solution. As used herein, the term “protein:cfDNAcomplexes” refers to protein:DNA complexes that are ex situ, i.e., notin a cell or nucleus. Protein:cfDNA complexes include but are notlimited to, DNA wrapped around or associated with nuclear proteins suchas nucleosomes (DNA wrapped around core of histone proteins),chromatosomes, transcription factors, or other nuclear proteins. In someembodiments a sample comprising protein:cfDNA complexes can be bloodserum, blood plasma, urine or other bodily fluids.

In some embodiments, a sample comprising protein:cfDNA complexes iscontacted with a solid phase. A solid phase or solid phase element canbe any solid phase that can associate with protein:cfDNA complexes orcan be functionalized to associate with protein:cfDNA complexes. In someembodiments a solid phase is a microplate or a bead.

In certain embodiments, the surface of the solid phase is functionalizedso as to bind protein. In some embodiments, the solid phase iscarboxylated. A solid phase can be functionalized with other suitablereagents able to bind to protein. In certain embodiments, the solidphase is a carboxylated bead or a carboxylated microplate. Nativespatially proximal nucleic acid molecules in the form of protein:cfDNAco-bound to a solid phase element are crosslinked. In certainembodiments, protein:cfDNA complexes bound to a carboxylated solid phaseare crosslinked to spatially and proximally bound protein:cfDNAcomplexes by contacting with a crosslinking reagent. In certainembodiments, a crosslinking reagent is formaldehyde. Other suitablecrosslinking reagents can be utilized. In some embodiments, nativespatially proximal nucleic acid molecules in the form of crosslinkedprotein:cfDNA co-bound to a solid phase element are released from thesolid phase (e.g., by an amide hydrolysis reaction).

FIG. 7 shows protein:cfDNA complexes after binding to a carboxylatedsolid phase element and undergoing protein:DNA crosslinking. In anembodiment, co-bound circulating protein:cfDNA complexes from bloodplasma are immobilized to a carboxylated surface, which binds proteins(e.g. COOH—NH2), and then crosslinked to each other to hold nSPNAs(natively occurring spatially proximal nucleic acid molecules) in closespatial proximity. Binding to a solid phase element prior tocrosslinking mitigates the chance of non-nSPNAs crosslinking to eachother by randomly colliding in solution if standard crosslinking wasperformed. This approach also allows the washing away of free-floatingcfDNA that is not bound to protein, because carboxylated surfaces onlybind proteins.

In certain embodiments, protein:cfDNA complexes are associated with asolid phase coated with a crosslinking reagent and are crosslinked tothe solid phase. In some embodiments, the crosslinking reagent ispsoralen. Other nucleic acid crosslinking agents can be utilized.

FIG. 8 shows protein:cfDNA complexes after crosslinking to a solid phaseelement coated in a nucleic acid crosslinking agent. In an embodiment,co-bound protein:cfDNA complexes from blood plasma are immobilized to asolid phase element via nucleic acid crosslinking, which binds DNA ofthe protein:cfDNA complexes to its surface. Crosslinking mediated bybinding to a solid phase mitigates the chance of non-nSPNAs crosslinkingto each other by random chance in solution if standard crosslinking wasperformed. This approach also allows the removal of proteins, since theproteins can be degraded (e.g. Proteinase K treatment), while leavingthe DNA bound to the solid phase element.

In some embodiments, the protein:cfDNA complexes, either crosslinked toone another or crosslinked to a solid phase are contacted with one ormore reagents that preserve spatial-proximal contiguity information inthe cell free DNA of the sample.

In certain embodiments, the one or more reagents that preservespatial-proximal contiguity information comprise reagents that generateproximity ligated nucleic acid molecules. In some embodiments, anaffinity purification marker is incorporated into the crosslinkedprotein:cfDNA complexes. In certain embodiments, the affinitypurification marker is a biotinylated nucleotide. In some embodiments,the spatially proximal cell free DNA of the protein:cfDNA complexes isligated to produce ligation products. In some embodiments, the ligationproducts are isolated. In some embodiments, the ligation products areisolated by affinity purification. In some embodiments, the affinitypurification is biotin:streptavidin enrichment of ligation junctions. Incertain embodiments, the ligation products do not contain an affinitypurification marker (e.g., a biotinylated nucleotide) and are isolatedby size selection. Size selection includes, but is not limited to, SPRIbeads and gel purification. In certain embodiments, reagents thatgenerate proximity ligated nucleic acid molecules comprise one or moreof at least one restriction endonuclease, a DNA polymerase, a pluralityof nucleotides comprising at least one biotinylated nucleotide, and aligase. In some embodiments, there are two restriction endonucleases.

FIG. 9 shows capturing spatial-proximal contiguity information viaproximity ligation from protein:cfDNA complexes. In an embodiment of themethod, (i) native spatially proximal nucleic acids (nSPNAs) from acell-free nucleic acids source (e.g. blood plasma), are co-bound to asolid phase element and crosslinked. This is followed by (ii) proximityligation to generate ligation products (LPs), which may have an affinitypurification marker at the ligation junction (e.g. an incorporatedbiotinylated nucleotide). While HiC is depicted, PL methods aresub-classified as 3C-based and HiC-based and there are many specificvariations of PL (as described herein). In HiC (iv), the cell-freenucleic acid ends are marked (e.g. biotinylated) and then ligated tocreate marked ligated products (MLPs, MLPs are a manifestation of LPs),bearing an affinity purification marker at the LJs. After MLPgeneration, affinity purification is used to enrich for MLPs comprisingLJs and such fragments are prepared as nucleic acid templates and areready for sequencing—i.e. the nucleic acids from the MLPs that containat least an LJ are enriched and prepared as a template and sequenced inHiC, to deplete uMLPs (unligated MLPs that do not usually manifest LJs).

Proximity ligation can also be utilized to preserve spatial-proximalcontiguity information when protein:cfDNA complexes are crosslinked to asolid phase.

In certain embodiments, the one or more reagents that preservespatial-proximal contiguity information comprise a Tn5 tetramer (see Laiet al. Nature Methods. Vol. 15, September 2018, 741-747). In someembodiments, the Tn5 tetramer has a biotinylated linker sequence. Insome embodiments, the marked Tn5-mediated loops are isolated by affinitypurification. In some embodiments, the affinity purification isbiotin:streptavidin enrichment of the marked Tn5-mediated loops. Incertain embodiments, Tn5-mediated loops are isolated by size selection.Size selection includes, but is not limited to, SPRI beads and gelpurification.

FIG. 10 shows capturing spatial-proximal contiguity information viaTn5-mediated looping from protein:cfDNA complexes bound to a solidphase. In an embodiment, (i) native spatially proximal nucleic acids(nSPNAs) from a cell-free nucleic acids source (e.g. blood plasma), areco-bound to a solid phase element and crosslinked. Followed by (ii)introduction of a Tn5 tetramer, comprising two Tn5 dimers linked by alinker sequence comprising inward facing mosaic end (ME) sequences. Then(iii), tagmentation by the Tn5 tetramer on both co-bound protein:cfDNAcomplexes creates an oligonucleotide link between the nSPNAs, withbiotin marking the successful co-tagmentation events. Finally (iv), oncefragments are separated from Tn5, affinity purification is used toenrich for marked Tn5-mediated loops and fragments prepared as nucleicacid templates and are ready for sequencing.

FIG. 11 shows capturing spatial-proximal contiguity information viaTn5-mediated looping from protein:cfDNA complexes crosslinked to a solidphase element. In an embodiment, (i) native spatially proximal nucleicacids (nSPNAs) from a cell-free nucleic acids source (e.g. bloodplasma), are crosslinked to a solid phase element. This is followed by(ii) degrading of proteins, leaving cfDNA crosslinked to the solid phaseelement. Then (iii) introduction of a Tn5 tetramer, comprising two Tn5dimers linked by a linker sequence comprising inward facing mosaic end(ME) sequences. Then (iv), tagmentation by the Tn5 tetramer on bothco-crosslinked cfDNA molecules creates an oligonucleotide link betweenthe nSPNAs, with biotin marking the successful co-tagmentation events.Finally (v), affinity purification is used to enrich for markedTn5-mediated loops and such fragments are prepared as nucleic acidtemplates and are ready for sequencing.

In some embodiments, the one or more reagents that preservespatial-proximal contiguity information in the cell free DNA of thesample are used with methods in which the native spatially proximalnucleic acids (nSPNAs) from a cell-free nucleic acids source (e.g. bloodplasma), are bound to a solid phase element (not crosslinked to thesolid phase element). Reagents may include one or more of at least onerestriction endonuclease, a DNA polymerase, a plurality of nucleotidescomprising at least one biotinylated nucleotide, a ligase and a Tn5tetramer with a biotinylated linker sequence.

In some embodiments, the one or more reagents that preservespatial-proximal contiguity information comprise reagents thatcompartmentalize the cell—free DNA protein complexes released from asolid support and tag the compartmentalized cell-free DNA proteincomplexes with a compartment-specific barcoded oligonucleotide. In someembodiments, tagging is by ligating on a compartment-specific barcodedsequencing adaptor. In some embodiments, tagging is by annealing andextending using a compartment specific barcoded primer. In someembodiments, tagging is by a transposome carrying a compartment-specificmolecular barcode. Reagents may include one or more of a liquid dropletgenerator, a barcoded oligonucleotide, a ligase, a DNA polymerase, andnucleotides.

FIG. 12 shows capturing spatial-proximal contiguity information fromprotein:cfDNA complexes via compartmentalization and tagging withcompartment-specific molecular barcodes. In an embodiment, (i) nativespatially proximal nucleic acids (nSPNAs) from a cell-free nucleic acidssource (e.g. blood plasma), are co-bound to a solid phase element andcrosslinked. Followed by (ii) detachment of the crosslinkedprotein:cfDNA complex from the solid phase element. Then (iii)compartmentalization of the crosslinked protein:cfDNA complexes andintroducing a compartment-specific molecular barcode, such as ligating acompartment-specific barcoded oligonucleotide. Finally (v), barcodedtemplate molecules are purified and prepared as nucleic acid templatesand are ready for sequencing, whereby the molecular barcode is themolecular identifier for which nSPNAs were spatially proximal.

In some embodiments, the one or more reagents that preservespatial-proximal contiguity information comprise reagents that affinitypurify the protein:cfDNA complexes, compartmentalize the affinitypurified protein:cfDNA complexes and tag the compartmentalizedprotein:cfDNA complexes with a compartment specific molecular barcode.In some embodiments, tagging is by ligating on a compartment-specificbarcoded sequencing adaptor. In some embodiments, tagging is byannealing a compartment specific barcoded PCR primer and extending theprimer. In some embodiments, tagging is by a transposome carrying acompartment-specific molecular barcode.

FIG. 13 shows capturing spatial-proximal contiguity information fromprotein:cfDNA complexes via compartmentalization with solid phaseelement and tagging with compartment-specific molecular barcodes. In anembodiment, (i) native spatially proximal nucleic acids (nSPNAs) from acell-free nucleic acids source (e.g. blood plasma), are co-bound to asolid phase element and crosslinked. This is followed by (ii) detachmentof the crosslinked protein:cfDNA complex from the solid phase element.Then (iii) affinity purification, such as with streptavidin coatedbeads, followed by compartmentalization of the crosslinked protein:cfDNAcomplexes bound to the solid phase element whereby acompartment-specific molecular barcode is tagged to the cfDNA. Finally(iv), barcoded template molecules are purified and prepared as nucleicacid templates and are ready for sequencing, whereby the molecularbarcode is the molecular identifier for which nSPNAs were spatiallyproximal.

In some embodiments, the one or more reagents that preservespatial-proximal contiguity information comprise Tn5 bound to a solidphase. In some embodiments, the solid phase is a bead. In certainembodiments, the Tn5 comprises a virtual compartment-specific molecularbarcode.

FIG. 14 shows capturing spatial-proximal contiguity information fromprotein:cfDNA complexes via virtual compartmentalization usingbead-linked transposome carrying a virtual compartment-specificmolecular barcode. In an embodiment, (i) native spatially proximalnucleic acids (nSPNAs) from a cell-free nucleic acids source (e.g. bloodplasma), are co-bound to a solid phase element and crosslinked. This isfollowed by (ii) detachment of the crosslinked protein:cfDNA complexfrom the solid phase element. Then (iii) tagmentation with bead-linkedtransposome carrying unique molecular barcodes, thereby creating virtualcompartments. Finally (iv), barcoded template molecules are purified andprepared as nucleic acid templates and are ready for sequencing, wherebythe molecular barcode is the molecular identifier for which nSPNAs werespatially proximal.

Data Analysis/Applications

Nucleic acids from FFPE samples, deeply formalin-fixed samples orsamples comprising protein:cfDNA complexes prepared by methods describedherein, in conjunction with proximity ligation methods (e.g., HiC, 3C,4C, 5C) or other methods that capture spatial-proximal contiguityinformation generate contiguity-preserved sequencing data forapplications, such as haplotype phasing and genomic rearrangementdetection For example, Selvaraj et al. BMC Genomics (2015), Selvaraj etal, Nature Biotechnol (2013), and PCT/US2014/047243 described HiC datafor haplotype phasing and Engreitz et al. (PLOS ONE September2012/Volume 7/Issue 9/e44196) has described HiC data for genomicrearrangement analysis in human disease. Several other papers havedescribed using HiC data for genomic rearrangement detection (Dixon etal. Nature Genetics (2018); Chakraborty and Ay, Bioinformatics (2018);Harewood et al. Genome Biology (2017). One such analysis tool forrearrangement detection is HiC-Breakfinder tool(https://github.com/dixonlab/hic_breakfinder) from Dixon et al, NatureGenetics, 2018. For example, as described in Example 21 FFPE-HiC datacan be used be used to identify a translocation in samples exhibiting arange of mutant allele frequencies (50% to 10%) and at differentsequencing depths (15×, 5× and 1×). Also, as described in Example 22,translocations can be detected by applying Capture-HiC to processed FFPEsamples for which a breakpoint is not captured by a capture sequencingprobe. Other contiguity-preservation-enabled analyses and applicationsinclude, but are not limited to, de novo genome and metagenome assembly,structural variation detection, and others.

Experiments demonstrating the value of the described protocols forpreparing FFPE tumor tissue samples in conjunction with HiC for thediscovery of translocations in tumor tissue samples are described inExample 23 and Example 24.

Methylation

In some embodiments the nucleic acids with preserved spatial-proximalcontiguity information generated by the methods described herein arecontacted with a bisulfite reagent prior to PCR and sequencing to enablethe concurrent analysis of spatial proximity and DNA methylation at baseresolution. In some embodiments the bisulfite reagent is sodiumbisulfite.

In some embodiments HiC ligation products are generated using a HiCprotocol as previously described (Rao et al, Cell, 2014, Li et al,Biorxiv 2018). DNA is sheared to an approximate length of 400 bp, andligation junction are enriched using streptavidin beads. Illuminalibrary construction ensues while the DNA is attached to thestreptavidin bead, as previously described (Rao et al, Cell, 2014).Directly after adapter ligation, DNA is subject to bisulfite conversion,using methods known in the art. Unmethylated lambda DNA is spiked in at0.5% prior to bisulfite conversion in order to estimate the conversionrate. The bisulfite converted DNA is purified, amplified, and sequenced.

In some embodiments sheared HiC ligation products are treated with abisulfite reagent and purified (Stamenova, Biorxiv, 2018). Ligationjunctions are then enriched using streptavidin beads. DNA is thendetached from the beads, and prepared as a sequencing library usingtechniques known in the art for converting ssDNA into a dsDNA sequencinglibrary. Adapter ligated molecules are then subject to libraryamplification and sequencing.

Methods of bisulfite conversion or derivations of these methods can beapplied to nucleic acids molecules comprising spatial-proximalcontiguity information obtained from FFPE, deeply fixed, orprotein:cfDNA samples using the methods described herein (e.g.,proximity ligation, solid substrate-mediated proximity capture (SSPC),compartmentalization and tagging an and use of a Tn5 tetramer).

After sequencing, methods known to the art can be used to analyze thedata in the context of spatial-proximity and long-range sequencecontiguity, such as but not limited to using the spatial proximalcontiguity information to inform genome folding patterns(Lieberman-Aiden, Science, 2009), and genomic rearrangement analysis(Dixon et al, Nature Genetics, 2018). Similarly, methods known to theart can also be applied to analyze the DNA methylation status (Lister etal, Nature, 2009; Shultz et al, Nature, 2015). Additionally, methodsknown in the art can also be applied to concurrently analyze the DNAmethylation status with respect to 3D genome folding (Li et al, Biorxiv2018; Stamenova, Biorxiv, 2018), revealing DNA chemical modificationsproperties and DNA folding patterns in parallel. Specifically in thecontext of applying this method to protein:cfDNA complexes, it is wellknown in the art that DNA methylation status of cell free nucleic acidscan inform tissue of origin analyses as well as several other cfDNAanalysis, including but not limited to the non-invasive detection oftumor DNA, prenatal diagnoses, and organ transplantation monitoring(Zeng et al, Journal of Genetics and Genomics, 2018; Lehmann-Werman etal, PNAS, 2016). Moreover, similar to DNA methylation status, DNAfolding patterns are also tissue-type specific, and thus HiC signalobtained from cfDNA:protein complexes may also aid in such non-invasivecfDNA analyses such as cancer diagnoses and organ transplantationmonitoring. Also, because it is known that HiC signal uniquely captureslong-range sequence contiguity information to significantly enhancegenomic rearrangement analyses (Dixon et al, Nature Genetics, 2018), HiCapplied to cfDNA:protein complexes could enrich for such genomicrearrangement signal from liquid biopsy samples and greatly benefitearly non-invasive cancer diagnoses. And finally, the combination andconcurrent analysis of both DNA methylation and DNA spatial proximityand long-range contiguity will synergize to better enable the analysesdescribed herein.

Kits

In some embodiments, provided are kits for carrying out methodsdescribed herein. Kits often comprise one or more containers thatcontain one or more components described herein. A kit comprises one ormore components in any number of separate containers, packets, tubes,vials, multiwell plates and the like, or components may be combined invarious combinations in such containers. Kit components and reagents areas described herein.

One or more of the following components, for example, may be included ina kit: (i) one or more dewaxing reagents (e.g., xylene, mineral oil,etc); (ii) one or more lysis buffers (e.g., lysis buffer with one ormore salts, a protease inhibitor and a non-ionic, non-denaturingdetergent, etc); (iii) one or more denaturing detergents (e.g., sodiumdodeccyl sulfate, etc.); (iv) one or more reagents to quench adenaturing detergent (e.g.TritonX-100, etc.), (v) one or moreextracellular matrix proteases (e.g., a collagenase and/or a dispase,ColI, ColIII, ColIV, Dispase I, etc.); (vi) one or more reagents thatpreserve spatial-proximal contiguity information and (vii) printedmatter (e.g. directions, labels, etc). In some embodiments, a kitcomprises one or more reagents that preserve spatial-proximal contiguityinformation that generate proximity ligated nucleic acid molecules(e.g., a restriction endonuclease or two restriction endonucleases, aDNA polymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase, etc.). In some embodiments, a kitcomprises one or more reagents that preserve spatial-proximal contiguityinformation comprising solid phase elements (e.g., beads, solid phasesubstrates functionalized with a nucleic acid crosslinking reagent, suchas psoralen, solid phase substrates functionalized with an affinitypurification molecule, such as streptavidin, and an affinitypurification marker, such as biotin, solid phase substratesfunctionalized with a transposase, such as Tn5, comprising a barcodedoligonucleotide. In some embodiments, a kit comprises one or morereagents that preserve spatial-proximal contiguity informationcomprising compartmentalization reagents and compartment-specificmolecular barcodes. In some embodiments, a kit comprises one or morereagents that preserve spatial-proximal contiguity informationcomprising a Tn5 tetramer (e.g., a Tn5 tetramer that comprises abiotinylated linker sequence). In some embodiments, a kit comprises asurface on which the methods described herein are carried out, in wholeor part, (e.g., a pathology slide, etc.).

In some embodiments, a kit does not include one or more dewaxingreagents.

One or more of the following components, for example, may be included ina kit: (i) a solid phase and (ii) one or more reagents that preservespatial-proximal contiguity information. In some embodiments, a kitcomprises a solid phase that comprises a carboxylated surface (e.g., amicroplate, a bead, etc.). In some embodiments, a kit comprises a solidphase (e.g., a magnetic bead, etc.) that is coated with a cross-linkingreagent (e.g., psoralen, etc.). In some embodiments, a kit comprises oneor more reagents that preserve spatial-proximal contiguity informationthat generate proximity-ligated nucleic acid molecules (e.g., arestriction endonuclease or two restriction endonucleases, a DNApolymerase, a plurality of nucleotides sometimes comprising at least onebiotinylated nucleotide, and a ligase, etc.). In some embodiments, a kitcomprises compartmentalization reagents (e.g., a microfluidiccompartmentalization device that produces microfluidic droplets ormicrotiter plate wells into which complexes are diluted),compartment-specific molecular barcodes and one or more reagents toattach barcodes to preserve spatial-proximal contiguity information(e.g., reagents for primer extension polymerization (PEP), reagents forligation or a transposase comprising a barcoded oligonucleotide).

In some embodiments, a kit comprises one or more reagents to affinitypurify native spatially proximal nucleic acids in the form ofprotein:cfDNA complexes prior to compartmentalization (e.g., an affinitypurification marker such as a biotinylated nucleotide and an affinitypurification molecule such as streptavidin). In some embodiments, a kitcomprises one or more reagents that preserve spatial-proximal contiguityinformation comprising a Tn5 tetramer (e.g., a Tn5 tetramer thatcomprises a biotinylated linker sequence). In some embodiments, a kitcomprises one or more reagents that preserve spatial-proximal contiguityinformation comprise a bead-linked transposome (e.g., Tn5, etc.)comprising a compartment-specific barcoded oligonucleotide.

In some embodiments, a kit comprises a bisulfite reagent. In someembodiments, the bisulfite reagent is sodium bisulfite.

A kit sometimes is utilized in conjunction with a process, and caninclude instructions for performing one or more processes and/or adescription of one or more compositions. A kit may be utilized to carryout a process described herein. Instructions and/or descriptions may bein tangible form (e.g., paper and the like) or electronic form (e.g.,computer readable file on a tangle medium (e.g., compact disc) and thelike) and may be included in a kit insert. A kit also may include awritten description of an internet location that provides suchinstructions or descriptions.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology.

Example 1: Preparation of Formalin-Fixed Paraffin Embedded (FFPE)Samples De-Waxing and Re-Hydration

-   -   1. Collect FFPE tissue sections (e.g., 5-10 um in thickness)        from an FFPE block or FFPE slide. Transfer FFPE section to clean        1.5 ml tube.    -   2. Add 1 mL of xylene solution and incubate for 10 min. at RT.    -   3. Centrifuge at 17,860×g (15,000 rpm) for 5 min. at RT    -   4. Resuspend the deparaffinized tissue in 1 mL of 100% ethanol        and incubate for 10 min. at RT    -   5. Centrifuge at 17,860×g for 5 min. at RT    -   6. Resuspend the deparaffinized tissue in 1 mL of water and        incubate for 10 min. at RT    -   7. Centrifuge at 17,860×g for 5 min. at RT

ECM Protease Dissociation (Optional)

Example tissue dissociation buffers (all diluted in 1× PBS, pH=7.4):

Buffer 1: 0.05% Collagenase III

Buffer 2: 0.05% Collagenase IV

Buffer 3: 0.025% Collagenase IV+0.025% Dispase I (Neutral Protease I)

Buffer 4: 0.1% Collagenase I+0.025% Dispase I (Neutral Protease I)

Buffer 5: 0.05% Dispase I (Neutral Protease)

-   -   8. Add 1 mL of enzymatic tissue dissociation buffer (see above)        and incubate for ˜20 min at 37° C.    -   9. Centrifuge at 1,000×g for 10 min at RT and remove        supernatant.    -   10. Add 200 uL of lysis buffer as described herein and incubate        at 4° C. for 20 min and vortex every 4 minutes throughout the        incubation.    -   11. Spin down at 1000×g and remove supernatant.    -   12 Resuspend sample in 1× Tris.    -   13. Add SDS to a final concentration of 0.5% and incubate at        74° C. for 40 min.    -   14. Add TritonX-100 to a final 10:1 v/v concentration relative        to SDS, incubate at 37° C. for 15 min.

Prepare proximity ligation products, solid substrate-mediated proximitycapture (SSPC) products, compartmentalized and tagged products or Tn5tetramer products for use in DNA sequencing as described herein.

Example 2: Alternative Method for Preparation of Formalin-FixedParaffin-Embedded (FFPE) Samples (No De-Waxing and Rehydrating)

-   -   1. Collect FFPE tissue sections (e.g., 5-10 um in thickness)        from an FFPE block or FFPE slide. Transfer FFPE section to clean        1.5 m1 tube.    -   2. Add 200 uL of lysis buffer as described herein and incubate        at 4° C. for 20 min and vortex every 4 minutes throughout the        incubation.    -   3. Spin down at 1000×g and decant supernatant.    -   4. Resuspend sample in 1× Tris.    -   5. Add SDS to a final concentration of 0.5% and incubate at        74° C. for 40 min.    -   6. Add TritonX-100 to a final 10:1 v/v concentration relative to        SDS, incubate at 37° C. for 15 min.

Prepare proximity ligation products, solid substrate-mediated proximitycapture (SSPC) products, compartmentalized and tagged products or Tn5tetramer products for use in DNA sequencing as described herein.

Example 3: FFPE—De-Waxing/Rehydration, No Cell Lysis, SDS Step,with/without Enzymatic Tissue Dissociation De-Waxing and Re-Hydration

-   -   1. Collect FFPE tissue sections (e.g. 5-10 um in thickness) from        an FFPE block or FFPE slide. Transfer FFPE section to clean 1.5        ml tube.    -   2. Add 1 mL of xylene solution and incubate for 10 min. at RT.    -   3. Centrifuge at 17,860×g (15,000 rpm) for 5 min. at RT    -   4. Resuspend the deparaffinized tissue in 1 mL of 100% ethanol        and incubate for 10 min. at RT    -   5. Centrifuge at 17,860×g for 5 min. at RT    -   6. Resuspend the deparaffinized tissue in 1 mL of water and        incubate for 10 min. at RT    -   7. Centrifuge at 17,860×g for 5 min. at RT

ECM Protease Dissociation (Optional)

Example tissue dissociation buffers (all diluted in 1× PBS, pH=7.4):

Buffer 1: 0.05% Collagenase III

Buffer 2: 0.05% Collagenase IV

Buffer 3: 0.025% Collagenase IV+0.025% Dispase I (Neutral Protease I)

Buffer 4: 0.1% Collagenase I+0.025% Dispase I (Neutral Protease I)

Buffer 5: 0.05% Dispase I (Neutral Protease)

-   -   8. Add 1 mL of enzymatic tissue dissociation buffer (see above)        and incubate for ˜20 min at 37° C.    -   9. Centrifuge at 1,000×g for 10 min at RT and remove        supernatant.    -   10. Resuspend sample in 1× Tris.    -   11. Add SDS to a final concentration of 0.5% and incubate at        74° C. for 40 min.    -   12. Add TritonX-100 to a final 10:1 v/v concentration relative        to SDS, incubate at 37° C. for 15 min.

Prepare proximity ligation products, solid substrate-mediated proximitycapture (SSPC) products, compartmentalized and tagged products or Tn5tetramer products for use in DNA sequencing as described herein.

Example 4-FFPE—(without De-Waxing/Rehydration), No Cell Lysis, SDS Step,No Enzymatic Tissue Dissociation

-   -   1. Collect FFPE tissue sections (e.g. 5-10 um in thickness) from        an FFPE block or FFPE slide. Transfer FFPE section to clean 1.5        ml tube.    -   2. Resuspend sample in 1× Tris.    -   3. Add SDS to a final concentration of 0.5% and incubate at        74° C. for 40 min.    -   4. Add TritonX-100 to a final 10:1 v/v concentration relative to        SDS, incubate at 37° C. for 15 min.

Prepare proximity ligation products, solid substrate-mediated proximitycapture (SSPC) products, compartmentalized and tagged products or Tn5tetramer products for use in DNA sequencing as described herein.

Example 5: Deeply Formalin-Fixed Sample (Pulverize, with/without EnzymeDissociation, Lysis, SDS)

-   -   1. Pulverize tissue on dry ice with liquid nitrogen, mortar and        pestle.    -   2. Transfer tissue to clean 1.5 mL microcentrifuge tube.

ECM Protease Dissociation (Optional)

Example tissue dissociation buffers (all diluted in 1× PBS, pH=7.4):

Buffer 1: 0.05% Collagenase III

Buffer 2: 0.05% Collagenase IV

Buffer 3: 0.025% Collagenase IV+0.025% Dispase I (Neutral Protease I)

Buffer 4: 0.1% Collagenase I+0.025% Dispase I (Neutral Protease I)

Buffer 5: 0.05% Dispase I (Neutral Protease)

-   -   3. Add 1 mL of enzymatic tissue dissociation buffer (see above)        and incubate for ˜20 min at 37° C.    -   4. Centrifuge at 1,000×g for 10 min at RT and remove        supernatant.    -   5. Add 200 uL of lysis buffer as described herein and incubate        at 4° C. for 20 min and vortex every 4 minutes throughout the        incubation.    -   6. Spin down at 1000×g and decant supernatant.    -   7. Resuspend sample in 1× Tris.    -   8. Add SDS to a final concentration of 0.5% and incubate at        74° C. for 40 min.    -   9. Add TritonX-100 to a final 10:1 v/v concentration relative to        SDS, incubate at 37° C. for 15 min.

Prepare proximity ligation products, solid substrate-mediated proximitycapture (SSPC) products, compartmentalized and tagged products or Tn5tetramer products for use in DNA sequencing as described herein.

Example 6: Protein:cfDNA Spatial-Proximal Contiguity Preservation PlasmaIsolation:

-   -   1. Collect blood into blood collection tube (BCT).    -   2. Isolate plasma, containing protein/cfDNA complexes.

Crosslinking Methods:

Using a Carboxylated Surface

-   -   1. Bind the proteins of the protein:cfDNA complexes to a        carboxylated surface, such as a carboxylated bead or        carboxylated microplate surface.    -   2. Wash away free-floating cfDNA, leaving behind only        protein:cfDNA complexes bound to the carboxylated surface.    -   3. Crosslink the protein to cfDNA, such as using formaldehyde.

Using a Solid Phase Element Coated with a Crosslinking Reagent

-   -   1. Crosslink protein:cfDNA complexes to a solid phase element        coated in a crosslinking reagent, e.g., a magnetic bead coated        in a nucleic acid crosslinking reagent, such as psoralen.    -   2. Optional: Degrade proteins, such as using Proteinase K,        leaving the cfDNA crosslinked to the solid phase element.

If Capturing Spatial-Proximal Contiguity Information via ProximityLigation:

-   -   1. Blunt ends of cfDNA        -   a. Optionally incorporate an affinity purification marker,            such as a biotinylated nucleotide.    -   2. Ligate spatially proximal cfDNA, producing ligation products.    -   3. Isolate ligation products by either:        -   a. Affinity purification (e.g. biotin:streptavidin            enrichment of the ligation junctions)        -   b. Size selection—unligated cfDNA fragments are 100-240 bp,            so any proximally ligated cfDNA molecules must be >240bp and            can be purified via SPRI beads, gel purification, etc.

If Capturing Spatial-Proximal Contiguity Information via Tn5 Tetramer:

-   -   1. Add Tn5 tetramer to tagment the spatially proximal cfDNA,        creating a physical link (or “loop”) between two spatially        proximal cfDNA fragments via an oligonucleotide linker sequence        and thereby producing Tn5-mediating loop products.        -   a. Optionally use a biotinylated linker sequence.    -   2. Isolate spatially linked products by either:        -   a. Affinity purification (e.g. biotin:streptavidin            enrichment of the biotinylated linker)        -   b. Size selection—typical cfDNA fragments are 100-240 bp, so            any Tn5 mediating loop products must be >240 bp and can be            purified via SPRI beads, gel purification, etc.            If Capturing Spatial-Proximal Contiguity Information via            Compartmentalizing the Crosslinked Protein:cfDNA Complexes            (without a Solid Phase Element) and Tagging with a            Compartment Specific Molecular Barcode:    -   1. Release (i.e. unbind) crosslinked protein:cfDNA complexes,        such as from the carboxylated surface.    -   2. Physically compartmentalize protein:cfDNA complexes, such as        in a liquid droplet.    -   3. Tag cfDNA within each compartment with a compartment-specific        molecular barcode        -   a. For example, ligating a compartment-specific barcoded            sequencing adapter or annealing a compartment specific            barcoded PCR primer.            If Capturing Spatial-Proximal Contiguity Information via            Compartmentalizing the Crosslinked Protein:cfDNA Complexes            (with a Solid Phase Elements) and Tagging with a Compartment            Specific Molecular Barcode:    -   1. Blunt ends of cfDNA and incorporate an affinity purification        marker, such as a biotinylated nucleotide.    -   2. Release (i.e. unbind) crosslinked protein:cfDNA complexes,        such as from the carboxylated surface.    -   3. Affinity purify protein:cfDNA complexes, for example using        streptavidin-coated beads.    -   4. Physically compartmentalize protein:cfDNA complexes bound to        a solid phase element, such as in a liquid droplet.    -   5. Tag cfDNA within each compartment with a compartment-specific        molecular barcode        -   a. For example, ligating a compartment-specific barcoded            sequencing adapter or annealing a compartment specific            barcoded PCR primer.            If Capturing Spatial-Proximal Information via Virtually            Compartmentalizing the Cross Linked Protein:cfDNA Complexes            by Tagging with a Unique Molecular Barcode, Mediated by Tn5            Bound to a Solid Phase Element (a “Bead-Linked            Transposome”):    -   1. Release (i.e. unbind) crosslinked protein:cfDNA complexes,        such as from the carboxylated surface.    -   2. Add uniquely barcoded bead-linked transposomes.    -   3. Tag cfDNA from the same protein:cfDNA complex with the same        molecular barcode using tagmentation from the bead-linked        transposome.

Example 7: Extension of the Chromatin Solubilization and DecompactionReaction Improves Spatial-Proximal Contiguity Signal from FFPE Samples

LPs were prepared from 10 um FFPE sections with a combination of two4-cutter restriction enzymes, but solubilized and decompacted thechromatin prior to digestion using the published time of 10 minutes ofSDS treatment, or extended treatments of 40 minutes, in replicate. OnceLPs were generated, the protocol continued with fragmentation, biotinenrichment, and preparation as a template and short-read sequencing. Thefraction of sequencing readouts that are long-cis was used as a proxyfor the preservation of spatial-proximal contiguity. The published SDStreatment time of 10 min contained only ˜1.65% of templates that arelong-cis, while 40 minutes of SDS treatment increased this fraction to˜7.75%, nearly a 5-fold increase (see FIG. 1A), yet still belowstate-of-the-art levels of capturing spatial-proximal contiguity (˜40%long-cis readouts). These results indicate that the spatial-proximalcontiguity signal can be improved by chromatin solubility anddecompaction optimization.

Example 8: Extension of the Chromatin Solubilization and DecompactionReaction to 80 min Improves Spatial-Proximal Contiguity Signal from FFPESamples

LPs were prepared from 10 um FFPE sections using a combination of two4-cutter restriction enzymes, but solubilized and decompacted thechromatin prior to digestion using previously optimized 40 min (See FIG.1A), or extended treatment durations of 60 or 80 minutes, in replicate.Once LPs were generated, the protocol continued with fragmentation,biotin enrichment, and preparation as a template and short-readsequencing. The fraction of sequencing readouts that are long-cis wasused as a proxy for the preservation of spatial-proximal contiguity. Theprevious extended SDS treatment time of 40 min contained only ˜18% oftemplates that are long-cis, while 60 minutes of SDS treatment increasedthis fraction to ˜25%, and 80 minutes of SDS treatment increased to ˜37%long-cis (see FIG. 1B). The long-cis from 80 min SDS duration indicatesa >20-fold increase relative to 10 min of SDS (See FIG. 1A), yet stillbelow state-of-the-art levels of capturing spatial-proximal contiguity(˜40% long-cis readouts). These results indicate that thespatial-proximal contiguity signal can be improved by chromatinsolubility and decompaction optimization.

Example 9: Extension of the Chromatin Solubilization and DecompactionReaction to 180 min Improves Spatial-Proximal Contiguity Signal from 10um FFPE Samples

LPs were prepared from 10 um FFPE sections using a combination of two4-cutter restriction enzymes, but solubilized and decompacted thechromatin prior to digestion using 40 min, or extended treatmentdurations of 80, 120, or 180 minutes, in replicate. Once LPs weregenerated, the protocol continued with fragmentation, biotin enrichment,and preparation as a template and short-read sequencing. The fraction ofsequencing readouts that are long-cis was used as a proxy for thepreservation of spatial-proximal contiguity. The SDS treatment time of40 min contained only ˜16% of templates that are long-cis, while 80,120, and 180 minutes of SDS treatment increased this fraction to ˜16%,28%, and 37%, on average (see FIG. 10). The long-cis from 180 min SDSduration indicates a >20-fold increase relative to 10 min of SDS (SeeFIG. 1A), yet still below state-of-the-art levels of capturingspatial-proximal contiguity (˜40% long-cis readouts).

Example 10: Extension of the Chromatin Solubilization and DecompactionReaction to 180 min does not Significantly Improve Spatial-ProximalContiguity Signal from 5 um FFPE Samples

LPs were prepared from 5 um FFPE sections using a combination of two4-cutter restriction enzymes, but solubilized and decompacted thechromatin prior to digestion using 40 min, or extended treatmentdurations of 80, 120, or 180 minutes, in replicate. Once LPs weregenerated, the protocol continued with fragmentation, biotin enrichment,and preparation as a template and short-read sequencing. The fraction ofsequencing readouts that are long-cis was used as a proxy for thepreservation of spatial-proximal contiguity. SDS treatment time of 40min contained only ˜26% of templates that are long-cis, while 80, 120,and 180 minutes of SDS treatment increased this fraction to ˜30.5%, 31%,and 32%, on average (see FIG. 1D). While the long-cis is still asignificant improvement relative to 10 min of SDS (See FIG. 1D), itstill below state-of-the-art levels of capturing spatial-proximalcontiguity (˜40% long-cis readouts). These results indicate that for 5um sections, there is no tangible benefit of extending the SDS reactionbeyond 80 min, and it appears the SDS duration optimization plateausbelow desirable levels of long-cis readouts.

Example 11: Increasing the temperature of chromatin solubilization anddecompaction to extreme heat surprisingly improves spatial-proximalcontiguity signal from 5 um FFPE samples.

To explore the effect of temperature and duration of the chromatinsolubilization and decompaction reaction, LPs were prepared from 5 umFFPE sections using a combination of two 4-cutter restriction enzymes,but solubilized and decompacted the chromatin prior to digestion using10, 40, or 80 minutes at 50° C., 62° C., or 74° C., as indicated. OnceLPs were generated, the protocol continued with fragmentation, biotinenrichment, and preparation as a template and short-read sequencing. Thefraction of sequencing readouts that are long-cis was used as a proxyfor the preservation of spatial-proximal contiguity. 50° C. SDStreatments from 10-80 min resulted in ˜2.8-10.2% long-cis templates,while 62° C. SDS treatments from 10-80 min resulted in ˜6.8-39% long-cistemplates. Surprisingly, 74° C. SDS treatments from 10-40 min resultedin ˜31.7-54.2% long-cis templates (see FIG. 2A), a significantimprovement over conventional 62° C. treatments reaching superior levelsof spatial-proximal contiguity signal. This result is surprising becauseextremely high heat can begin to denature AT-rich dsDNA and reverse theformalin crosslinks that are critical to hold together the 3D chromatinstructure and required for capturing spatial-proximal contiguity viaproximity ligation or other methods that entail crosslinking.

Example 12: Increasing the Temperature of Chromatin Solubilization andDecompaction to Extreme Heat Surprisingly Improves Spatial-ProximalContiguity Signal from 10 um FFPE Samples

To determine whether the extreme heat during chromatin solubilizationand decompaction reaction improves long-cis in thicker (10 um) FFPEsection, we prepared LPs from 10 um FFPE sections using a combination oftwo 4-cutter restriction enzymes, but solubilized and decompacted thechromatin prior to digestion using 10, 40, or 80 minutes at 74° C., inreplicate. Once LPs were generated, the protocol continued withfragmentation, biotin enrichment, and preparation as a template andshort-read sequencing. The fraction of sequencing readouts that arelong-cis was used as a proxy for the preservation of spatial-proximalcontiguity. 74° C. SDS treatments for 10 min resulted in virtually no(˜0.5%) long-cis templates, while 40 and 80 min resulted in ˜53.5% and55.5% long-cis templates, on average, respectively (see FIG. 2B). Thissurprising result also indicates the robustness of the 74° C. for 40 minprotocol to 5 and 10 um FFPE samples.

Example 13: Chromatin Solubilization and Decompaction at 74° C. for 40min Optimally Captures Spatial-Proximal Contiguity Signal from aClinical Human FFPE Tumor Sample

To examine whether the extreme heat during chromatin solubilization anddecompaction reaction improves long-cis in a patient-derived FFPE tumorsample, LPs were prepared from 7-8 um FFPE sections, including acellular lysis step and using a combination of two 4-cutter restrictionenzymes, but solubilized the chromatin prior to digestion using 40, or80 minutes at 62° C. or 74° C., in replicate, as indicated. 62° C. for80 minutes was selected because 80 minutes was the time at which furtherextension yielded no improvement in long-cis on 5 um sections, whereas74° C. for 40 min was selected based on the optimal performance androbustness at various section thickness (FIGS. 2A and 2B). Once LPs weregenerated, the protocol continued with fragmentation, biotin enrichment,and preparation as a template and short-read sequencing. The fraction ofsequencing readouts that are long-cis was used as a proxy for thepreservation of spatial-proximal contiguity. 62° C. SDS treatment for 80min resulted in 38% long-cis templates, whereas 74° C. SDS treatmentsfor 40 min resulted in 53.5% long-cis templates (FIG. 3). Thissurprising result indicates the optimal capture of spatial-proximalcontiguity and robustness of the 74° C. for 40 min protocol inpatient-derived FFPE tissue.

Example 14: Cellular Lysis is not Required to Capture OptimalSpatial-Proximal Contiguity Signal from FFPE Samples

To examine whether the extreme heat during chromatin solubilization anddecompaction reaction eliminates the need for cellular lysis for FFPEsamples, LPs were prepared from 5 um FFPE sections by either including acellular lysis step per the optimized protocol described herein (seeFIGS. 1 to 3), or, without cellular lysis. A combination of two 4-cutterrestriction enzymes was used, but solubilized and decompacted thechromatin prior to digestion using 40 minutes at 74° C., in replicate.Once LPs were generated, the protocol continued with fragmentation,biotin enrichment, and preparation as a template and short-readsequencing. The fraction of sequencing readouts that are long-cis wasused as a proxy for the preservation of spatial-proximal contiguity.FFPE samples that underwent cellular lysis resulted in 59% long-cistemplates, whereas FFPE samples that had foregone cellular lysisresulted in 57.5% long-cis templates, on average (see FIG. 4). Thissurprising result indicates the optimal capture of spatial-proximalcontiguity and robustness of the 74° C. for 40 min protocol, which alsoobviates the need for cellular lysis.

Example 15: De-Waxing and Rehydration is Required to CaptureSpatial-Proximal Contiguity Signal from FFPE Samples when the SDSReaction is at 62° C.

To examine whether one can capture spatial-proximal contiguity signalfrom FFPE samples without having to de-wax and rehydrate the tissue, LPswere prepared from 10 um FFPE sections by either including standardde-waxing and rehydration per the optimized protocol described herein(see FIGS. 1-4), or, without subjecting the FFPE samples to de-waxing orrehydration (contrary to the standard procedures used fornext-generation sequencing from FFPE samples). A combination of two4-cutter restriction enzymes was used, but solubilized and decompactedthe chromatin prior to digestion using 10 or 40 minutes at 62° C., inreplicate. Once LPs were generated, the protocol continued withfragmentation, biotin enrichment, and preparation as a template andshort-read sequencing. The fraction of sequencing readouts that arelong-cis was used as a proxy for the preservation of spatial-proximalcontiguity. FFPE samples that were not dewaxed and rehydrated resultedin 0.8-1.4% long-cis templates, whereas FFPE samples that did receivede-waxing and rehydration resulted in 1.5-8.5% long-cis templates, onaverage, with a greater fraction of long-cis templates at longer SDSreaction durations (see FIG. 5A). These results indicate that thecapture of spatial-proximal contiguity signal is minimal when the FFPEsample forgoes de-waxing and rehydration and a 62° C. chromatinsolubilization and decompaction reaction.

Example 16: De-Waxing and Rehydration is not Required to Capture OptimalSpatial-Proximal Contiguity Signal from FFPE Samples when the SDSReaction is at 74° C. for 40 min

To examine whether one can capture optimal spatial-proximal contiguitysignal from FFPE samples without having to de-wax and rehydrate thetissue, LPs were prepared from 5 and 10 um FFPE sections by eitherincluding standard de-waxing and rehydration per the optimized protocoldescribed herein (see FIGS. 1-4), or, without subjecting the FFPEsamples to de-waxing or rehydration (contrary to the standard procedureused for next-generation sequencing from FFPE samples). A combination oftwo 4-cutter restriction enzymes was used, but solubilized anddecompacted the chromatin prior to digestion using SDS for 40 minutes at74° C., in replicate. Once LPs were generated, the protocol continuedwith fragmentation, biotin enrichment, and preparation as a template andshort-read sequencing. The fraction of sequencing readouts that arelong-cis was used as a proxy for the preservation of spatial-proximalcontiguity. FFPE samples that were not dewaxed and rehydrated resultedin 53-60% long-cis templates, whereas FFPE samples that did receivede-waxing and rehydration resulted in 52.4-59% long-cis templates, onaverage (see FIG. 5B). These results surprisingly indicate the optimalcapture of spatial-proximal contiguity from FFPE samples that foregode-waxing and rehydration and undergo a 74° C. chromatin solubilizationand decompaction reaction.

Example 17: Enzymatic Dissociation of Extracellular Matrix ProteinsImproves User-Friendliness without Compromising Optimal Capture ofSpatial-Proximal Contiguity Signal from FFPE Samples

To examine whether one can enzymatically dissociate the FFPE samples toimprove user-friendliness (e.g. improve ease of pipetting) withoutsacrificing the optimal capture of spatial-proximal contiguity, LPs wereprepared from 5 and 10 um FFPE sections by either including an enzymaticdissociation step (with Collagenase III, Collagenase IV, Collagenase I,Dispase, or a combination as indicated), or, without enzymaticdissociation per the optimized protocols described herein (see FIGS.1-5). A combination of two 4-cutter restriction enzymes, but solubilizedand decompacted the chromatin prior to digestion using SDS for 40minutes at 74° C., in replicate. Once LPs were generated, the protocolcontinued with fragmentation, biotin enrichment, and preparation as atemplate and short-read sequencing. The fraction of sequencing readoutsthat are long-cis was used as a proxy for the preservation ofspatial-proximal contiguity. All FFPE samples resulted in 50-60%long-cis templates, on average (see FIG. 6). These results indicate theoptimal capture of spatial-proximal contiguity from FFPE samples evenwhen the samples are pre-treated with an ECM protease to improveuser-friendliness of the workflow.

Example 18: Failure of Chromatin Digestion in FFPE Cells Processed usingStandard Protocol for Crosslinked Cells

10 um FFPE sections were prepared from human cell lines. The sample wasdewaxed and rehydrated and then processed using the initial steps of astandard HiC protocol for mammalian cells (treatment with lysis bufferfollowed by 10 min of solubilization and decompaction at 62° C.) andthen digestion using 1000 U of a 4-cutter restriction enzyme (DpnII). Anexcess of restriction enzyme was used to anticipate the overly fixedchromatin resulting from the FFPE sample. As a control, frozen fixedcells (not FFPE) were subject to standard HiC protocol for mammaliancells, with just 50 U of DpnII. After digestion the crosslinks werereversed and DNA was purified and the size was analyzed by gelelectrophoresis and Agilent TapeStation instrument (AgilentTechnologies, Inc., Santa Clara, Calif.). The gel electrophoresisanalysis indicated efficiently digested chromatin for the frozen cellsand virtually no digestion for the FFPE cells, despite having 20-foldmore restriction enzyme units (See FIG. 18). The TapeStation analysis ofthe average fragment size (see “Frag Size (bp)” row) supports the gelanalysis and provides more precise quantitation of the average size ofthe DNA after digestion.

Example 19: Automated HiC on FFPE Tissues

5 um FFPE sections of mouse liver tissue were de-waxed and rehydrated,and then put through a protocol optimized for FFPE samples, as describedherein (no tissue dissociation (extracellular matrix protease),treatment with lysis buffer and 40 min of solubilization anddecompaction at 74° C.) and subject to HiC using with 2 4-cutterrestriction enzymes. However the entire HiC protocol, from lysis throughreverse crosslinking and DNA purification was carried out on the AgilentBravo automated liquid handling platform (Agilent Technologies, Inc.,Santa Clara, Calif.). LPs were sheared using the Bioruptor® Picoinstrument (Diagenode, Danville, N.J.) and then DNA was returned to theBravo for automated library prep using KAPA HyperPrep (KAPABIOSYSTEMS,Capetown, South Africa). Library amplification was carried out in a PCRmachine, and the post-PCR amplicons were returned to the Bravo automatedliquid handling platform for purification. Upon shallow sequencing andanalysis, consistently high long-range cis readouts from each of the 8replicates analyzed in the automated experiment was observed (see FIG.19).

Example 20: Rapid Reverse Crosslinking in FFPE Tissues

5 um FFPE sections were put through a HiC protocol. Mouse liver tissuewas de-waxed and rehydrated, and then put through a protocol optimizedfor FFPE samples, as described herein (no tissue dissociation(extracellular matrix protease), treatment with lysis buffer and 40 minof solubilization and decompaction at 74° C.). The sample was thensubject to HiC using 2 4-cutter restriction enzymes. However the reversecrosslinking procedure was varied such that the tissue received thestandard treatment (30 min of Proteinase K (ProK)at 55° C. then 90 minat 68 C), just the lower temperature treatment (30 min of ProK at 55°C.), or no reverse crosslinking at all as a negative control. Shallowsequencing and analysis indicated high long-range cis readouts from thereduced reverse crosslinking protocol, comparable to the full reversecrosslinking protocol, and significantly better than no reversecrosslinking (see FIG. 20). Also, see Example 17 and FIG. 6, whichreversed crosslinking using the 30 min of ProK at 55° C. protocol.

In another set of experiments, the reverse crosslinking procedure wasincubation at 95° C. for 60 minutes in the absence of Proteinase K. Theamount of DNA that was purified was approximately equivalent to theamount obtained using the full reverse crosslinking protocol. The longcis values were approximately 48-50% long-range cis, comparable tovalues obtained using the full reverse crosslinking protocol.

Example 21: Highly Sensitive Discovery of a Known Translocation as aFunction of Mutant Allele Frequency and Sequencing Depth usingGenome-Wide FFPE-HiC Data

FFPE blocks of cells harboring a heterozygous a ROS1-SLC34A2translocation in every cell (50% mutant allele frequency (MAF)) or akaryotypically normal cell line (GM24385) were obtained from HorizonDiscovery (Waterbeach, United Kingdom). 10 um FFPE sections werede-waxed, rehydrated, treated with lysis buffer followed by 40 min ofsolubilization and decompaction at 62° C. and subject to HiC usingdigestion with 2 4-cutter restriction enzymes. Following deep sequencinganalysis (˜30×) on each sample, raw reads were computationally mixed atthe following cancer:normal ratios: 100:0; 80:20; 60:40, 40:60, 20:80,and 0:100, thus representing 50%, 40%, 30%, 20%, 10%, and 0% mutantallele frequency of the ROS1:SLC34A2 translocation (rows). This readmixing was also conducted in such a way that the total depth would befixed at either 15×, 5×, or 1× (columns). The translocation was thenidentified using HiC-Breakfinder which found the ROS1-SLC34A2translocation in all depth and MAF combination except the 10% MAF at 1×depth, and all the negative control conditions (0% MAF). Black arrowsare overlaid on the HiC contact maps around the ROS1-SLC34A2 genes incases which HiC-Breakfinder made the translocation call (see FIG. 21).

Example 22: Highly Sensitive Discovery of a Known Translocation Enabledby Targeted FFPE-HiC Data

10 um FFPE sections of cells harboring a heterozygous a ROS1-SLC34A2translocation in every cell (50% mutant allele frequency (MAF)) werede-waxed, rehydrated, treated with lysis buffer followed by 40 min ofsolubilization and decompaction at 62° C. and subject to HiC usingdigestion with 2 4-cutter restriction enzymes. Following deep sequencinganalysis (˜30×) on each sample, the effect of targeted HiC(“Capture-HiC”) was simulated by capturing all the HiC signal derivedfrom one locus in the genome ˜50 kb downstream from the trueROS1-SLC34A2 breakpoint. The HiC signal produced at only 30× depth(which is significantly less than typically oncology gene panelsequencing) was plotted across the top. Virtually zero HiC signal wasobserved upstream of the breakpoint on SLC34A2. The HiC signal wasobserved to peak at the breakpoint in SLC34A2 and then decrease for locimoving downstream from SLC34A2 (see FIG. 22). The location of thebreakpoint is in SLC34A2, is based on the peak HiC signal (andunderlying sequence information) and distance dependent decay signalmoving away from (in this case downstream) from the breakpoint.

Example 23: Discovery and Validation of Translocations in an FFPE GISTTumor

A 7-8 um FFPE section of a gastrointestinal stromal tumor (GIST) wasde-waxed and rehydrated, and then put through a protocol optimized forFFPE samples, as described herein (no tissue dissociation (extracellularmatrix protease), treatment with lysis buffer and 40 min ofsolubilization and decompaction at 74° C.). The sample was then subjectto HiC using 2 4-cutter restriction enzymes. Shallow sequencing analysisindicated high long-range cis readouts (see FIG. 23A). Furthermore,genome-wide shallow sequencing analysis (0.75× sequencing; ˜$30 costs)identified 19 translocations, with several breakpoints implicating genesassociated with cancer but not commonly targeted by gene panels. Forexample, the analysis discovered a POLA2-PIGU translocation. POLA2 hasat least 10 reported partners in cancer databases (i.e. COSMIC, Quiver,TCGA), and is recurrently translocated in GIST. PIGU has >13 reportedpartners, but neither POLA2 nor PIGU are targeted by tumor geneticprofiling panels (such as Agilent ClearSeq (Agilent Technologies, Inc.,Santa Clara, Calif.), underscoring the power of HiC to discoverpromiscuous and partner-agnostic translocations. To obtain sub-Kbbreakpoint resolution for PCR validation, sequencing was performed up to˜10× (see FIG. 23B). One inter-chr translocation involving UQCC1 (>12reported partners) and ARHGAP20 (1 reported translocation in B-CLL) wereselected for PCR validation. Test primer pairs (and control primerpairs, not shown) were designed to amplify across the translocationbreakpoint (see FIG. 23C). PCR results confirmed a single PCR amplicon(see FIG. 23D), validating the optimized FFPE-HiC protocol andtranslocation discovery analyses in FFPE tumor tissue.

Example 24: Discovery of Translocations in an FFPE Pediatric EpendymomaTumor

A 5 um FFPE section of a posterior fossa ependymoma (PFE) tumor wasde-waxed and rehydrated, and then put through a protocol optimized forFFPE samples, as described herein (no tissue dissociation (extracellularmatrix protease), treatment with lysis buffer and 40 min ofsolubilization and decompaction at 74° C.). The sample was then subjectto HiC using 2 4-cutter restriction enzymes. Genome-wide shallowsequencing analysis (0.25× sequencing; ˜$10 costs) revealed the presenceof several translocations (see FIG. 24B), similar to what has beenpreviously observed in HiC data from PFE cell lines (see FIG. 24A;personal communication with Dr. Lukas Chavez, UCSD). In fact, FFPE-HiCanalyses of this PFE tumor identified a chr1;chr3 translocation (seeFIG. 24C), whereby the chr1 partner was the same gene involved in achr1;chr8 translocation previously identified from a PFE cell linederived from a different individual, indicating the potential forFFPE-HiC to identify previously underappreciated, partner-agnostic, andrecurring translocations in tumor tissues. Also of note, this FFPE PFEtumor had previously been analyzed using conventional WGS and RNA-seq,but no somatic mutations or translocations were found, thus underscoringthe analytical sensitivity of FFPE-HiC compared to state-of-the-artgenome-wide (WGS) and targeted (RNA-seq) methodologies for translocationanalyses.

Example 25: Discovery of Translocations in FFPE Tumors Across ArchivalPeriods

5 um sections from FFPE blocks containing tumor tissue from eitherstomach, lung, thyroid, or liver were obtained from BioChain InstituteInc. (Newark, N.J.). Each tumor block had an archival period of 4, 5,10, or 18 years, respectively (see FIG. 25A). FFPE sections werede-waxed and rehydrated, and then put through a protocol optimized forFFPE samples, as described herein (no tissue dissociation (extracellularmatrix protease), treatment with lysis buffer and 40 min ofsolubilization and decompaction at 74° C.). The processed sections werethen subject to HiC using 2 4-cutter restriction enzymes Shallowsequencing analysis indicated moderately high, yet consistent long-rangecis readouts across the range of archival periods, even out to 18 years(see FIG. 25A) without negative impact on the quality of the long-rangeinformation in the data. From this extremely low sequencing depth (0.05×sequencing; ˜$2 costs) in the lung tumor, evidence of 7 translocationswere identified, including intra-chr translocations (see FIG. 25B),inter-chromosomal translocation between chr3;chr18 (see FIG. 25C) andchr3;chr7 (see FIG. 25D).

Example 26: High Quality FFPE-HiC from Low Input FFPE Tissue

5-10 um FFPE sections were obtained from mouse liver tissue or humanGIST, PFE, lung, liver, stomach, and thyroid tumors. Each tumor sectionwas de-waxed and rehydrated, and then put through a protocol optimizedfor FFPE samples, as described herein (no tissue dissociation(extracellular matrix protease), treatment with lysis buffer and 40 minof solubilization and decompaction at 74° C.). The processed sectionswere then subject to HiC using 2 4-cutter restriction enzymes Shallowsequencing analysis indicated moderate to high long-range cis readouts,even in cases where the total amount of DNA extraction from the FFPEtissue section was considered low input, defined here as <200 ng.

Example 27: Non-Limiting Examples of Embodiments

Listed hereafter are non-limiting examples of certain embodiments of thetechnology.

A1. A method for preparing nucleic acids from a formalin-fixedparaffin-embedded (FFPE) sample, that preserves spatial-proximalcontiguity information, comprising:

a) providing a formalin-fixed paraffin-embedded sample;

b) de-waxing the sample to produce a de-waxed sample;

c) rehydrating the de-waxed sample, thereby generating ade-waxed/rehydrated sample;

d) contacting the de-waxed/rehydrated sample with lysis buffer; therebygenerating a lysed sample;

e) contacting the lysed sample; with a denaturing detergent at atemperature greater than 65° C., thereby generating a solubilized anddecompacted sample; and

f) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

A1.1. The method of embodiment A1, wherein the formalin-fixedparaffin-embedded (FFPE) sample is a tissue sample.

A1.2. The method of embodiment A1 or A1.1, wherein thede-waxed/rehydrated sample is contacted with an extracellular matrixprotease prior to contact with lysis buffer.

A1.3. The method of embodiment A1.2, wherein the protease is acollagenase and/or a dispase.

A1.4. The method of embodiment A1.3, wherein the collagenase is ColI,ColIII or ColIV, and the dispase is Dispase I.

A2. The method of any one of embodiments A1 to A1.4, wherein contactwith the denaturing detergent is for greater than 10 minutes.

A3. The method of embodiment A2, wherein contact with the denaturingdetergent is 15 to 80 minutes.

A3.1. The method of embodiment A3, wherein contact with the denaturingdetergent is 30 to 50 minutes

A4. The method of embodiment A3.1, wherein contact with the denaturingdetergent about 40 minutes.

A4.1 The method of embodiment A4, wherein contact with the denaturingdetergent is 40 minutes.

A5. The method of any one of embodiments A1-A4.1, wherein thetemperature is greater than 65° C. and less than 80° C.

A6. The method of embodiment A5, wherein the temperature is between 70°C. and 80° C.

A7. The method of embodiment A6, wherein the temperature is about 74° C.

A7.1 The method of embodiment A7, wherein the temperature is 74° C.

A7.2. The method of any one of embodiments A1-A1.4, wherein contact witha denaturing detergent is for 40 minutes at a temperature of 74° C.

A8. The method of any one of embodiments A1-A7.2, wherein the detergentis sodium dodecyl sulfate (SDS).

A9. The method of any one of embodiments A1-A8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

A9.1. The method of embodiment A9, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

A9.2. The method of embodiment A9.1, comprising two restrictionendonucleases.

A10. The method of any one of embodiments A9 to A9.2, wherein theproximity ligated nucleic acid molecules are generated in situ.

A11. The method of any one of embodiments A1-A8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with the spatially proximalnucleic acid of the solubilized and decompacted sample to generatespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

A12. The method of embodiment A11, wherein the solid phase elements aresolid phase substrates functionalized with a nucleic acid crosslinkingagent.

A13. The method of embodiment A11, wherein the solid phase elements aresolid phase substrates functionalized with an affinity purificationmolecule and the nucleic acid molecules are labeled with an affinitypurification marker.

A13.1. The method of any one of embodiments A11 to A13, wherein thespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements is contacted with one or morereagents for compartmentalization and tagging with a molecular barcode.

A13.2. The method of embodiment A13.1, wherein the one or more reagentsfor compartmentalization comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

A13.3. The method of embodiment A13.1 or A13.2, wherein tagging with amolecular barcode is by primer extension polymerization (PEP) or byligation.

A14. The method of embodiment A11, wherein the solid phase elements aresolid phase substrates functionalized with a transposase comprising abarcoded oligonucleotide that generate spatially-proximal nucleic acidcomprising a barcoded oligonucleotide and complexed to the solid phasesubstrate functionalized with a transposase

A14.1. The method of embodiment A14, wherein the transposase is Tn5.

A15. The method of any one of embodiments A1 to A14.1, wherein theformalin-fixed paraffin-embedded sample is provided on a solid surface.

A15.1. The method of embodiment A15.1, wherein the solid surface is apathology slide.

A16. The method of any one of embodiments A1 to A8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and tag by attaching compartment-specificmolecular barcodes to the nucleic acids of the solubilized anddecompacted sample.

A16.1. The method of embodiment A16, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

A16.2. The method of embodiment A16 or A16.1, wherein the one or morereagents that tag by attaching compartment-specific molecular barcodescomprise reagents for primer extension polymerization (PEP), reagentsfor ligation or a transposase comprising a barcoded oligonucleotide.

A17. The method of any one of embodiments A1 to A16.2, wherein theformalin-fixed paraffin-embedded sample is provided as a tissue sectionof about 5 um to about 10 um in thickness.

A18. The method of any one of embodiments, A1 to A8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

A19. The method of any one of embodiments, A1 to A18, wherein step (f)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are subjected to bisulfite treatment to generatebisulfite treated nucleic acids with preserved spatial-proximalcontiguity information.

A20. The method of embodiment A19, wherein the bisulfite treated nucleicacids with preserved spatial-proximal contiguity information aresequenced to determine the methylation status of the nucleic acids withpreserved spatial-proximal contiguity information.

A21. The method of any one of embodiments A1 to A18, wherein step (f)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

A22. The method of embodiment A21, wherein the sequencing is at a depthof 30× or less.

A23. The method of embodiment A21 or A22, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

A24. The method of embodiment A23, wherein % of long-range cis readoutsis greater than 40% of the readouts.

A25. The method of any one of embodiments A1 to A18, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 4 years to about 20 years.

A26. The method of any one of embodiments A1 to A18, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 20 years to about 70 years.

A27. The method of embodiment A25 or A26, wherein step (f) generatesnucleic acids with preserved spatial-proximal contiguity information andthe nucleic acids with preserved spatial-proximal contiguity informationare sequenced to produce sequence readouts.

A28. The method of embodiment A27, wherein the sequencing at a depth of30× or less.

A29. The method of embodiment A27 or A28, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

A30. The method of any one of embodiments A1 to A18, wherein the nucleicacid obtained from the formalin-fixed paraffin-embedded (FFPE) sample isless than 200 ng.

A31. The method of embodiment A30, wherein step (f) generates nucleicacids with preserved spatial-proximal contiguity information and thenucleic acids with preserved spatial-proximal contiguity information aresequenced to produce sequence readouts.

A32. The method of embodiment A31, wherein the sequencing at a depth of30× or less.

A33. The method of embodiment A31 or A32, wherein the sequence read-outshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

A34. The method of embodiment A33, wherein % of long-range cis readoutsis greater than 40% of the readouts.

A35. The method of any one of embodiments A1 to A34, wherein the methodis essentially carried out using automated equipment.

A36. The method of any one of embodiments A1 to A35, wherein after step(f) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

A37. The method of embodiment A36, wherein the temperature is about 55°C.

A37.1. The method of embodiment A37, wherein the temperature is 55° C.

A38. The method of any one of embodiments A1 to A35, wherein after step(f) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

A38.1. The method of embodiment A38, wherein after step (f) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

B1. A method for preparing nucleic acids from a formalin-fixedparaffin-embedded (FFPE) sample, that preserves spatial-proximalcontiguity information, comprising:

a) providing a formalin-fixed paraffin-embedded sample;

b) de-waxing the sample to produce a de-waxed sample;

c) rehydrating the de-waxed sample, thereby generating ade-waxed/rehydrated sample;

d) contacting the de-waxed/rehydrated sample with a denaturing detergentat a temperature greater than 65° C., thereby generating a solubilizedand decompacted sample; and

e) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

B1.1. The method of embodiment B1, wherein the formalin-fixedparaffin-embedded (FFPE) sample is a tissue sample.

B1.2. The method of embodiment B1 or B1.1, wherein thedewaxed/rehydrated sample is contacted with an extracellular matrixprotease prior to contact with a denaturing detergent.

B1.3. The method of embodiment B1.2, wherein the protease is acollagenase and/or a dispase.

B1.4. The method of embodiment B1.3, wherein the collagenase is ColI,ColIII or ColIV and the dispase is Dispase I.

B2. The method of any one of embodiments B1 to B1.4, wherein contactwith the denaturing detergent is for greater than 10 minutes.

B3. The method of embodiment B2, wherein contact with the denaturingdetergent is 15 to 80 minutes.

B4. The method of embodiment B3, wherein contact with the denaturingdetergent is 30 to 50 minutes.

B4.1. The method of embodiment B4, wherein contact with the denaturingdetergent is about 40 minutes.

B4.2. The method of embodiment B4.1, wherein contact with the denaturingdetergent is 40 minutes.

B5. The method of any one of embodiments B1-B4.2, wherein thetemperature is greater than 65° C. and less than 80° C.

B6. The method of embodiment B5, wherein the temperature is between 70°C. and 80° C.

B7. The method of embodiment B6, wherein the temperature is about 74° C.

B7.1 The method of embodiment B7, wherein the temperature is 74° C.

B7.2. The method of any one of embodiments B1-B1.4, wherein contact witha denaturing detergent is for 40 minutes at a temperature of 74° C.

B8. The method of any one of embodiments B1-B7.2, wherein the detergentis sodium dodecyl sulfate (SDS).

B9. The method of any one of embodiments B1-B8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

B9.1. The method of embodiment B9, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

B9.2. The method of embodiment B9.1, comprising two restrictionendonucleases.

B10. The method of any one of embodiments B9 to B9.2, wherein theproximity ligated nucleic acid molecules are generated in situ.

B11. The method of any one of embodiments B1-B8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with the spatially-proximalnucleic acids of the solubilized and decompacted sample to generatespatially-proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

B12. The method of embodiment B11, wherein the solid phase elements aresolid phase substrates functionalized with a nucleic acid crosslinkingagent.

B13. The method of embodiment B11, wherein the solid phase elements aresolid phase substrates functionalized with an affinity purificationmolecule and the nucleic acid molecules are labeled with an affinitypurification marker.

B13.1. The method of any one of embodiments B11 to B13, wherein thespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements is contacted with one or morereagents for compartmentalization and tagging with a molecular barcode.

B13.2. The method of embodiment B13.1, wherein the one or more reagentsfor compartmentalization comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

B13.3. The method of embodiment B13.1 or B13.2, wherein tagging with amolecular barcode is by primer extension polymerization (PEP) or byligation.

B14. The method of embodiment B11, wherein the solid phase elements aresolid phase substrates functionalized with a transposase comprising abarcoded oligonucleotide that generate spatially-proximal nucleic acidcomprising a ligated barcoded oligonucleotide and complexed to the solidphase substrate functionalized with a transposase.

B14.1. The method of embodiment B14, wherein the transposase is Tn5.

B15. The method of any one of embodiments B1 to B14.1, wherein theformalin-fixed paraffin-embedded sample is provided on a solid surface.

B15.1. The method of embodiment B15, wherein the solid surface is apathology slide.

B16. The method of any one of embodiments B1 to B8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and attach compartment-specific molecularbarcodes to the nucleic acids of the solubilized and decompacted sample.

B16.1. The method of embodiment B16, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

B16.2. The method of embodiment B16 or B16.1, wherein the one or morereagents that tag by attaching compartment-specific molecular barcodescomprise reagents for primer extension polymerization (PEP), reagentsfor ligation or a transposase comprising a barcoded oligonucleotide.

B17. The method of any one of embodiments B1 to B16.2, wherein theformalin-fixed paraffin-embedded sample is provided as a tissue sectionof about 5 um to about 10 um in thickness.

B18. The method of embodiments B17, wherein the formalin-fixedparaffin-embedded sample is provided as a tissue section of about 5 umin thickness.

B19. The method of any one of embodiments, B1 to B8, wherein the one ormore reagents that preserve spatial-proximal contiguity informationcomprise a Tn5 tetramer.

B20. The method of any one of embodiments, B1 to B19, wherein step (e)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are subjected to bisulfite treatment to generatebisulfite treated nucleic acids with preserved spatial-proximalcontiguity information.

B21. The method of embodiment B20, wherein the bisulfite treated nucleicacids with preserved spatial-proximal contiguity information aresequenced to determine the methylation status of the nucleic acids withpreserved spatial-proximal contiguity information.

B22. The method of any one of embodiments B1 to B19, wherein step (e)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

B23. The method of embodiment B22, wherein the sequencing is at a depthof 30× or less.

B24. The method of embodiment B22 or B23, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

B25. The method of embodiment B24, wherein % of long-range cis readoutsis greater than 40% of the readouts.

B26. The method of any one of embodiments B1 to B19, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 4 years to about 20 years.

B27. The method of any one of embodiments B1 to B19, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 20 years to about 70 years.

B28. The method of embodiment B26 or B27, wherein step (e) generatesnucleic acids with preserved spatial-proximal contiguity information andthe nucleic acids with preserved spatial-proximal contiguity informationare sequenced to produce sequence readouts.

B29. The method of embodiment B28, wherein the sequencing at a depth of30× or less.

B30. The method of embodiment B28 or B29, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

B31. The method of any one of embodiments B1 to B19, wherein the nucleicacid obtained from the formalin-fixed paraffin-embedded (FFPE) sample isless than 200 ng.

B32. The method of embodiment B31, wherein step (e) generates nucleicacids with preserved spatial-proximal contiguity information and thenucleic acids with preserved spatial-proximal contiguity information aresequenced to produce sequence readouts.

B33. The method of embodiment B32, wherein the sequencing at a depth of30× or less.

B34. The method of embodiment B32 or B33, wherein the sequence read-outshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

B35. The method of embodiment B34, wherein % of long-range cis readoutsis greater than 40% of the readouts.

B36. The method of any one of embodiments B1 to B35, wherein the methodis essentially carried out using automated equipment.

B37. The method of any one of embodiments B1 to B36, wherein after step(e) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

B38. The method of embodiment B37, wherein the temperature is about 55°C.

B38.1. The method of embodiment B38, wherein the temperature is 55° C.

B39. The method of any one of embodiments B1 to B36, wherein after step(e) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

B39.1. The method of embodiment B39, wherein after step (e) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

C1. A method for preparing nucleic acids from a formalin-fixedparaffin-embedded (FFPE) sample that preserves spatial-proximalcontiguity information comprising:

a) providing a formalin-fixed paraffin-embedded sample, that has notbeen de-waxed/rehydrated;

b) contacting the formalin-fixed paraffin-embedded sample with lysisbuffer, thereby generating a lysed sample;

c) contacting the lysed sample with a denaturing detergent at atemperature greater than 65° C., thereby generating a solubilized anddecompacted sample; and

d) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

C1.1. The method of embodiment C1, wherein the formalin-fixedparaffin-embedded (FFPE) sample is a tissue sample.

C1.2. The method of embodiment C1 or C1.1, wherein thedewaxed/rehydrated sample is contacted with an extracellular matrixprotease prior to contact with the lysis buffer.

C1.3. The method of embodiment C1.2, wherein the protease is acollagenase and/or a dispase.

C1.4. The method of embodiment C1.3, wherein the collagenase is ColI,ColIII or ColIV and the dispase is Dispase I.

C2. The method of any one of embodiments C1 to C1.4, wherein contactwith the denaturing detergent is greater than 10 minutes.

C3. The method of embodiment C2, wherein contact with the denaturingdetergent is 15 to 80 minutes.

C3.1 The method of embodiment C3, wherein contact with the denaturingdetergent is 30 to 50 minutes.

C4. The method of embodiment C3.1, wherein contact with the denaturingdetergent is about 40 minutes.

C4.1. The method of embodiment C4, wherein contact with the denaturingis 40 minutes.

C5. The method of any one of embodiments C1 to C4.1, wherein thetemperature is greater than 65° C. and less than 80° C.

C6. The method of embodiment C5, wherein the temperature is between 70°C. and 80° C.

C7. The method of embodiment C6, wherein the temperature is about 74° C.

C7.1. The method of embodiment C7, wherein the temperature is 74° C.

C7.2. The method of any one of embodiments C1-C1.4, wherein contact withthe denaturing detergent is for 40 minutes at a temperature of 74° C.

C8. The method of any one of embodiments C1-C7.2, wherein the detergentis sodium dodecyl sulfate (SDS).

C9. The method of any one of embodiments C1-C8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

C9.1. The method of embodiment C9, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

C9.2. The method of embodiment C9.1, comprising two restrictionendonucleases.

C10. The method of any one of embodiments C9 to C9.2, wherein theproximity ligated nucleic acid molecules are generated in situ.

C11. The method of any one of embodiments C1-C8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with spatially proximal nucleicacids of the solubilized and decompacted sample to generate spatiallyproximal nucleic acid of the solubilized and decompacted samplecomplexed to solid phase elements.

C12. The method of embodiment C11, wherein the solid phase elements aresolid phase substrates functionalized with a nucleic acid crosslinkingagent.

C13. The method of embodiment C11, wherein the solid phase elements aresolid phase substrates functionalized with an affinity purificationmolecule and the nucleic acid molecules are labeled with an affinitypurification marker.

C13.1. The method of any one of embodiments C11 to C13, wherein thespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements is contacted with one or morereagents for compartmentalization and tagging with a molecular barcode.

C13.2. The method of embodiment C13.1, wherein the one or more reagentsfor compartmentalization comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

C13.3. The method of embodiment C13.1 or C13.2, wherein tagging with amolecular barcode is by primer extension polymerization (PEP) or byligation.

C14. The method of embodiment C11, wherein the solid phase elements aresolid phase substrates functionalized with a transposase comprising abarcoded oligonucleotide that generate spatially-proximal nucleic acidcomprising a ligated barcoded oligonucleotide and complexed to the solidphase substrate functionalized with a transposase

C14.1. The method of embodiment C14, wherein the transposase is Tn5.

C15. The method of any one of embodiments C1 to C14.1, wherein theformalin-fixed paraffin-embedded sample is provided on a solid surface.

C15.1. The method of embodiment C15, wherein the solid surface is apathology slide.

C16. The method of any one of embodiments C1 to C8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and attach compartment-specific molecularbarcodes to the nucleic acids of the solubilized and decompacted sample.

C16.1. The method of embodiment C16, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

C16.2. The method of embodiment C16 or C16.1, wherein the one or morereagents that tag by attaching compartment-specific molecular barcodescomprise reagents for primer extension polymerization (PEP), reagentsfor ligation or a transposase comprising a barcoded oligonucleotide.

C17. The method of any one of embodiments C1 to C16.2, wherein theformalin-fixed paraffin-embedded sample is provided as a tissue sectionof about 5 um to about 10 um in thickness.

C18. The method of any one of embodiments, C1 to C8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

C19. The method of any one of embodiments, C1 to C18, wherein step (d)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are subjected to bisulfite treatment to generatebisulfite treated nucleic acids with preserved spatial-proximalcontiguity information.

C20. The method of embodiment C19, wherein the bisulfite treated nucleicacids with preserved spatial-proximal contiguity information aresequenced to determine the methylation status of the nucleic acids withpreserved spatial-proximal contiguity information.

C21. The method of any one of embodiments C1 to C18, wherein step (d)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

C22. The method of embodiment C21, wherein the sequencing is at a depthof 30× or less.

C23. The method of embodiment C21 or C22, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

C24. The method of embodiment C23, wherein % of long-range cis readoutsis greater than 40% of the readouts.

C25. The method of any one of embodiments C1 to C18, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 4 years to about 20 years.

C26. The method of any one of embodiments C1 to C18, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 20 years to about 70 years.

C27. The method of embodiment C25 or C26, wherein step (d) generatesnucleic acids with preserved spatial-proximal contiguity information andthe nucleic acids with preserved spatial-proximal contiguity informationare sequenced to produce sequence readouts.

C28. The method of embodiment C27, wherein the sequencing at a depth of30× or less.

C29. The method of embodiments, C27 or C28, wherein the sequencereadouts have a % of long-range cis readouts greater than the % oflong-range cis readouts produced without contact with a denaturingdetergent at a temperature greater than 65° C.

C30. The method of any one of embodiments C1 to C18, wherein the nucleicacid obtained from the formalin-fixed paraffin-embedded (FFPE) sample isless than 200 ng.

C31. The method of embodiment C30, wherein step (d) generates nucleicacids with preserved spatial-proximal contiguity information and thenucleic acids with preserved spatial-proximal contiguity information aresequenced to produce sequence readouts.

C32. The method of embodiment C31, wherein the sequencing at a depth of30× or less.

C33. The method of embodiment C31 or C32, wherein the sequence read-outshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

C34. The method of embodiment C33, wherein % of long-range cis readoutsis greater than 40% of the readouts.

C35. The method of any one of embodiments C1 to C34, wherein the methodis essentially carried out using automated equipment.

C36. The method of any one of embodiments C1 to C35, wherein after step(d) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

C37. The method of embodiment C36, wherein the temperature is about 55°C.

C37.1. The method of embodiment C37, wherein the temperature is 55° C.

C38. The method of any one of embodiments C1 to C35, wherein after step(d) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

C38.1. The method of embodiment C38, wherein after step (d) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

D1. A method for preparing nucleic acids from a formalin-fixedparaffin-embedded (FFPE) sample that preserves spatial-proximalcontiguity information comprising:

a) providing a formalin-fixed paraffin-embedded sample, that has notbeen de-waxed/rehydrated;

b) contacting the formalin-fixed paraffin-embedded sample with adenaturing detergent at a temperature greater than 65° C., therebygenerating a solubilized and decompacted sample; and

c) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

D1.1. The method of embodiment D1, wherein the formalin-fixedparaffin-embedded (FFPE) sample is a tissue sample.

D1.2. The method of embodiment D1 or D1.1, wherein thedewaxed/rehydrated sample is contacted with an extracellular matrixprotease prior to contact with a denaturing detergent.

D1.3. The method of embodiment D1.2, wherein the protease is acollagenase and/or a dispase.

D1.4. The method of embodiment D1.3, wherein the collagenase is ColI,ColIII or ColIV and the dispase is Dispase I.

D2. The method of any one of embodiments D1, to D1.4, wherein contactwith the denaturing detergent is for greater than 10 minutes.

D3. The method of embodiment D2, wherein contact with the denaturingdetergent is 15 to 80 minutes.

D3.1. The method of embodiment D3, wherein contact with the denaturingdetergent is 30 to 50 minutes.

D4. The method of embodiment D3.1, wherein contact with the denaturingdetergent is about 40 minutes.

D4.1. The method of embodiment D4, wherein contact with the denaturingdetergent is 40 minutes.

D5. The method of any one of embodiments D1-D4.1, wherein thetemperature is greater than 65° C. and less than 80° C.

D6. The method of embodiment D5, wherein the temperature is between 70°C. and 80° C.

D7. The method of embodiment D6, wherein the temperature is about 74° C.

D7.1. The method of embodiment D7, wherein the temperature is 74° C.

D7.2. The method of any one of embodiments D1 to D1.4, wherein contactwith the denaturing detergent is for 40 minutes at a temperature of 74°C.

D8. The method of any one of embodiments D1-D7.2, wherein the detergentis sodium dodecyl sulfate (SDS).

D9. The method of any one of embodiments D1-D8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

D9.1. The method of embodiment D9, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of atleast one restriction endonuclease, a DNA polymerase, a plurality ofnucleotides comprising at least one biotinylated nucleotide, and aligase.

D9.2. The method of embodiment D9.1, comprising two restrictionendonucleases.

D10. The method of any one of embodiments D9 to D9.2, wherein theproximity ligated nucleic acid molecules are generated in situ.

D11. The method of any one of embodiments D1-D8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with spatially-proximal nucleicacids of the solubilized and decompacted sample to generatespatially-proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

D12. The method of embodiment D11, wherein the solid phase elements aresolid phase substrates functionalized with a nucleic acid crosslinkingagent.

D13. The method of embodiment D11, wherein the solid phase elements aresolid phase substrates functionalized with an affinity purificationmolecule and the nucleic acid molecules are labeled with an affinitypurification marker.

D13.1. The method of any one of embodiments D11 to D13, wherein thespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements is contacted with one or morereagents for compartmentalization and tagging with a molecular barcode.

D13.2. The method of embodiment D13.1, wherein the one or more reagentsfor compartmentalization comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

D13.3. The method of embodiment D13.1 or D13.2, wherein tagging with amolecular barcode is by primer extension polymerization (PEP) or byligation.

D14. The method of embodiment D11, wherein the solid phase elements aresolid phase substrates functionalized with a transposase comprising abarcoded oligonucleotide that generate spatially-proximal nucleic acidcomprising a ligated barcoded oligonucleotide and complexed to the solidphase substrate functionalized with a transposase.

D14.1. The method of embodiment D14, wherein the transposase is Tn5.

D15. The method of any one of embodiments D1 to D14.1, wherein theformalin-fixed paraffin-embedded sample is provided on a solid surface.

D15.1. The method of embodiment D15.1, wherein the solid surface is apathology slide.

D16. The method of any one of embodiments D1 to D8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and attach compartment-specific molecularbarcodes to the nucleic acids of the solubilized and decompacted sample.

D16.1. The method of embodiment D16, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

D16.2. The method of embodiment D16 or D16.1, wherein the one or morereagents that tag by attaching compartment-specific molecular barcodescomprise reagents for primer extension polymerization (PEP), reagentsfor ligation or a transposase comprising a barcoded oligonucleotide.

D17. The method of any one of embodiments D1 to D16.2, wherein theformalin-fixed paraffin-embedded sample is provided as a tissue sectionof about 5 um to about 10 um in thickness.

D18. The method of embodiments D17, wherein the formalin-fixedparaffin-embedded sample is provided as a tissue section of about 5 umin thickness.

D19. The method of any one of embodiments, D1 to D8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

D20. The method of any one of embodiments, D1 to D19, wherein step (c)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are subjected to bisulfite treatment to generatebisulfite treated nucleic acids with preserved spatial-proximalcontiguity information.

D21. The method of embodiment D20, wherein the bisulfite treated nucleicacids with preserved spatial-proximal contiguity information aresequenced to determine the methylation status of the nucleic acids withpreserved spatial-proximal contiguity information.

D22. The method of any one of embodiments D1 to D19, wherein step (c)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

D23. The method of embodiment D22, wherein the sequencing is at a depthof 30× or less.

D24. The method of embodiment D22 or D23, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

D25. The method of embodiment D24, wherein % of long-range cis readoutsis greater than 40% of the readouts.

D26. The method of any one of embodiments D1 to D19, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 4 years to about 20 years.

D27. The method of any one of embodiments D1 to D19, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 20 years to about 70 years.

D28. The method of embodiment D26 or D27, wherein step (c) generatesnucleic acids with preserved spatial-proximal contiguity information andthe nucleic acids with preserved spatial-proximal contiguity informationare sequenced to produce sequence readouts.

D29. The method of embodiment D28, wherein the sequencing at a depth of30× or less.

D30. The method of embodiment D28 or D29, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

D31. The method of any one of embodiments D1 to D19, wherein the nucleicacid obtained from the formalin-fixed paraffin-embedded (FFPE) sample isless than 200ng.

D32. The method of embodiment D31, wherein step (c) generates nucleicacids with preserved spatial-proximal contiguity information and thenucleic acids with preserved spatial-proximal contiguity information aresequenced to produce sequence readouts.

D33. The method of embodiment D32, wherein the sequencing at a depth of30× or less.

D34. The method of embodiment D32 or D33, wherein the sequence read-outshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

D35. The method of embodiment D34, wherein % of long-range cis readoutsis greater than 40% of the readouts.

D36. The method of any one of embodiments D1 to D35, wherein the methodis essentially carried out using automated equipment.

D37. The method of any one of embodiments D1 to D36, wherein after step(c) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

D37.1. The method of embodiment D37, wherein the temperature is about55° C.

D37.2. The method of embodiment D37.1, wherein the temperature is 55° C.

D38. The method of any one of embodiments D1 to D36, wherein after step(c) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

D38.1. The method of embodiment D38, wherein after step (c) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

E1. A method for preparing nucleic acids from a deeply formalin-fixedsample that preserves spatial-proximal contiguity informationcomprising:

a) providing a deeply formalin-fixed sample;

b) contacting the deeply formalin-fixed sample with lysis buffer,thereby generated a lysed sample;

c) contacting the lysed sample with a denaturing detergent at atemperature greater than 65° C., thereby generating a solubilized anddecompacted sample; and

d) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

E1.1. The method of embodiment E1, wherein the deeply formalin-fixedsample is a tissue sample.

E1.2. The method of embodiment E1 or E1.1, wherein the deeplyformalin-fixed sample is contacted with an extracellular matrix proteaseprior to contact with lysis buffer.

E1.3. The method of embodiment E1.2, wherein the protease is acollagenase and/or a dispase.

E1.4. The method of embodiment E1.3, wherein the collagenase is ColI,ColIII or ColIV and the dispase is Dispase I.

E1.5. The method of any one of embodiments E1 to E1.4, wherein thedeeply formalin-fixed sample is pulverized.

E2. The method of any one of embodiments E1 to E1.5, wherein contactwith the denaturing detergent is for greater than 10 minutes.

E3. The method of embodiment E2, wherein contact with the denaturingdetergent is 15 to 80 minutes.

E3.1. The method of embodiment E3, wherein contact with the denaturingdetergent is 30 to 50 minutes.

E4. The method of embodiment E3.1, wherein contact with the denaturingdetergent is about 40 minutes.

E4.1. The method of embodiment E4, wherein contact with the denaturingdetergent is 40 minutes.

E5. The method of any one of embodiments E1 to E4.1, wherein thetemperature is greater than 65° C. and less than 80° C.

E6. The method of embodiment E5, wherein the temperature is between 70°C. and 80° C.

E7. The method of embodiment E6, wherein the temperature is about 74° C.

E7.1. The method of embodiment E7, wherein the temperature is 74° C.

E7.2. The method of any one of embodiments E1-E1.5, wherein contact withthe denaturing detergent is for 40 minutes at a temperature of 74° C.

E8. The method of any one of embodiments E1 to E7.2, wherein thedetergent is sodium dodecyl sulfate (SDS).

E9. The method of any one of embodiments E1-E8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

E9.1. The method of embodiment E9, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

E9.2. The method of embodiment E9.1, comprising two restrictionendonucleases.

E10. The method of any one of embodiment E9 to E9.2, wherein theproximity ligated nucleic acid molecules are generated in situ.

E11. The method of any one of embodiments E1-E8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with spatially-proximal nucleicacids of the solubilized and decompacted sample to generatespatially-proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

E12. The method of embodiment E11, wherein the solid phase elements aresolid phase substrates functionalized with a nucleic acid crosslinkingagent.

E13. The method of embodiment E11, wherein the solid phase elements aresolid phase substrates functionalized with an affinity purificationmolecule and the nucleic acid molecules are labeled with an affinitypurification marker.

E13.1. The method of any one of embodiments E11 to E13, wherein thespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements is contacted with one or morereagents for compartmentalization and tagging with a molecular barcode.

E13.2. The method of embodiment E13.1, wherein the one or more reagentsfor compartmentalization comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

E13.3. The method of embodiment E13.1 or E13.2, wherein tagging with amolecular barcode is by primer extension polymerization (PEP) or byligation.

E14. The method of embodiment E11, wherein the solid phase elements aresolid phase substrates functionalized with a transposase comprising abarcoded oligonucleotide that generate spatially-proximal nucleic acidcomprising a ligated barcoded oligonucleotide that are complexed to thesolid phase substrate functionalized with a transposase

E14.1. The method of embodiment E14, wherein the transposase is Tn5.

E15. The method of any one of embodiments E1 to E14.1, wherein thedeeply formalin-fixed sample is provided on a solid surface.

E15.1. The method of embodiment E15.1, wherein the solid surface is apathology slide.

E16. The method of any one of embodiments E1 to E8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and attach compartment-specific molecularbarcodes to the nucleic acids of the solubilized and decompacted sample.

E16.1. The method of embodiment E16, wherein the one or more reagentsthat compartmentalize are a microfluidic compartmentalization devicethat produces microfluidic droplets or microtiter plate wells into whichcomplexes are diluted.

E16.2. The method of embodiment E16 or E16.1, wherein the one or morereagents that tag by attaching compartment-specific molecular barcodescomprise reagents for primer extension polymerization (PEP), reagentsfor ligation or a transposase comprising a barcoded oligonucleotide.

E17. The method of any one of embodiments, E1 to E8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

E18. The method of any one of embodiments, E1 to E17, wherein step (d)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are subjected to bisulfite treatment to generatebisulfite treated nucleic acids with preserved spatial-proximalcontiguity information.

E19. The method of embodiment E18, wherein the bisulfite treated nucleicacids with preserved spatial-proximal contiguity information aresequenced to determine the methylation status of the nucleic acids withpreserved spatial-proximal contiguity information.

E20. The method of any one of embodiments E1 to E17, wherein step (d)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

E21. The method of embodiment E20, wherein the sequencing is at a depthof 30× or less.

E22. The method of embodiment E20 or E21, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

E23. The method of embodiment E22, wherein % of long-range cis readoutsis greater than 40% of the readouts.

E24. The method of any one of embodiments E1 to E17, wherein the deeplyformalin-fixed sample has an archival period of about 4 years to about20 years.

E25. The method of any one of embodiments E1 to E17, wherein the deeplyformalin-fixed sample has an archival period of about 20 years to about70 years.

E26. The method of embodiment E24 or E25, wherein step (d) generatesnucleic acids with preserved spatial-proximal contiguity information andthe nucleic acids with preserved spatial-proximal contiguity informationare sequenced to produce sequence readouts.

E27. The method of embodiment E26, wherein the sequencing at a depth of30× or less.

E28. The method of embodiment E26 or E27, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

E29. The method of any one of embodiments E1 to E17, wherein the nucleicacid obtained from the deeply formalin-fixed sample is less than 200 ng.

E30. The method of embodiment E29, wherein step (d) generates nucleicacids with preserved spatial-proximal contiguity information and thenucleic acids with preserved spatial-proximal contiguity information aresequenced to produce sequence readouts.

E31. The method of embodiment E30, wherein the sequencing at a depth of30× or less.

E32. The method of embodiment E30 or E31, wherein the sequence read-outshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

E33. The method of embodiment E32, wherein % of long-range cis readoutsis greater than 40% of the readouts.

E34. The method of any one of embodiments E1 to E33, wherein the methodis essentially carried out using automated equipment.

E35. The method of any one of embodiments E1 to E34, wherein after step(d) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

E36. The method of embodiment E35, wherein the temperature is about 55°C.

E37. The method of embodiment E36, wherein the temperature is 55° C.

E38. The method of any one of embodiments E1 to E34, wherein after step(d) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

E38.1. The method of embodiment E38, wherein after step (d) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

F1. A method for preparing nucleic acids from a sample comprisingprotein:cfDNA complexes, that preserves spatial-proximal contiguityinformation, comprising:

a) providing a sample comprising protein:cfDNA complexes;

b) crosslinking the protein:cfDNA complexes to neighboring protein:cfDNAcomplexes; and

c) contacting the crosslinked protein:cfDNA complexes with one or morereagents that preserve spatial-proximal contiguity information in thecell free DNA of the sample.

F2. The method of embodiment F1, wherein the sample is blood serum,blood plasma or urine.

F2.1. The method of embodiment F1 or F2, wherein the protein:cfDNAcomplexes are nucleosome complexes or chromatosome complexes.

F2.2. The method of any one of embodiments F1 to F2.1, wherein theprotein:cfDNA complexes in the sample are contacted with a solid phaseprior to crosslinking, thereby generating protein:cfDNA complexesassociated with a solid phase.

F3. The method of embodiment F2.2, wherein the solid phase binds theprotein:cfDNA complexes.

F3.1. The method of embodiments F2.2 or F3, wherein the crosslinkingagent is formaldehyde.

F4. The method of any one of embodiments F1 to F3.1, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

F5. The method of embodiment F4, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of arestriction endonuclease, a DNA polymerase, a plurality of nucleotidescomprising at least one biotinylated nucleotide, and a ligase.

F6. The method of any one of embodiments F1 to F3.1, wherein the one ormore reagents that preserve spatial-proximal contiguity informationcomprise a Tn5 tetramer.

F7. The method of any one of embodiments F1 to F3.1, wherein theprotein:cfDNA complexes are released from the solid support and the oneor more reagents that preserve spatial-proximal contiguity informationcomprise reagents that compartmentalize the protein:cfDNA complexesreleased from the solid support and tag the compartmentalizedprotein:cfDNA complexes with a compartment specific molecular barcode.

F7.1. The method of embodiment F7, wherein the one or more reagents thatcompartmentalize comprise a microfluidic compartmentalization devicethat produces microfluidic droplets or microtiter plate wells into whichcomplexes are diluted.

F7.2. The method of embodiment F7 or F7.1, wherein the one or morereagents that tag the compartmentalized protein:cfDNA complexes with acompartment specific molecular barcode comprise reagents for primerextension polymerization (PEP), reagents for ligation or a transposasecomprising a barcoded oligonucleotide.

F8. The method of any one of embodiments F7 to F7.2, further comprisingone or more reagents to affinity purify the protein:cfDNA complexes.

F9. The method of any one of embodiments F1 to F3.1, wherein one or morereagents that preserve spatial-proximal contiguity information compriseTn5 bound to a solid phase.

F9.1. The method of embodiment F9, wherein the Tn5 comprises a virtualcompartment specific molecular barcode.

F10. The method of any one of embodiments, F1 to F9.1, wherein step (c)generates cell free DNA with preserved spatial-proximal contiguityinformation and the cell free DNA with preserved spatial-proximalcontiguity information is subjected to bisulfite treatment to generatebisulfite treated cell free DNA with preserved spatial-proximalcontiguity information.

F11. The method of embodiment F10, wherein the bisulfite treated cellfree DNA with preserved spatial-proximal contiguity information issequenced to determine the methylation status of the cell free DNA withpreserved spatial-proximal contiguity information.

F12. The method of any one of embodiments F1 to F9.1, wherein step (c)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

F13. The method of embodiment F12, wherein the sequencing is at a depthof 30× or less.

F14. The method of any one of embodiments F1 to F13, wherein the methodis essentially carried out using automated equipment.

F15. The method of any one of embodiments F1 to F14, wherein after step(c) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

F16. The method of embodiment F15, wherein the temperature is about 55°C.

F17. The method of embodiment F16, wherein the temperature is 55° C.

F18. The method of any one of embodiments F1 to F14, wherein after step(c) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

F19. The method of embodiment F18, wherein after step (c) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

G1. A method for preparing nucleic acid from a sample comprisingprotein:cfDNA complexes, that preserves spatial-proximal contiguityinformation, comprising:

a) providing a sample comprising protein:cfDNA complexes;

b) contacting the sample with a solid phase, thereby generatingprotein:cfDNA complexes associated with a solid phase;

c) cross-linking the protein:cfDNA complexes to neighboringprotein:cfDNA complexes or to the solid phase; and

d) contacting the crosslinked protein:cfDNA complexes with one or morereagents that preserve spatial-proximal contiguity information in thecell free DNA of the sample.

G2. The method of embodiment G1, wherein the sample is blood serum,blood plasma or urine.

G2.1. The method of embodiment G1 or G2, wherein the protein:cfDNAcomplexes are nucleosome complexes or chromatosome complexes.

G3. The method of any one of embodiments G1 to G2.1, wherein the solidphase binds the protein:cfDNA complexes and the protein:cfDNA complexesbound to the solid phase are contacted with a cross-linking reagent.

G3.1. The method of embodiment G3, wherein the crosslinking agent isformaldehyde.

G4. The method of any one of embodiments G1 to G2.1, wherein the solidphase is coated with a crosslinking agent and the protein:cfDNAcomplexes are crosslinked to the solid phase.

G4.1. The method of embodiment G4, wherein the crosslinking reagent ispsoralen.

G5. The method of any one of embodiments G1 to G4.1, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

G5.1. The method of embodiment G5, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of arestriction endonuclease, a DNA polymerase, a plurality of nucleotidescomprising at least one biotinylated nucleotide, and a ligase.

G6. The method of any one of embodiments G1 to G4.1, wherein the one ormore reagents that preserve spatial-proximal contiguity informationcomprise a Tn5 tetramer.

G7. The method of any one of embodiments G1 to G4.1, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize the protein:cfDNA complexes released fromthe solid support and tag the compartmentalized protein:cfDNA complexeswith a compartment specific molecular barcode.

G7.1. The method of embodiment G7, wherein the one or more reagents thatcompartmentalize comprise a microfluidic compartmentalization devicethat produces microfluidic droplets or microtiter plate wells into whichcomplexes are diluted.

G7.2. The method of embodiment G7 or G7.1, wherein the one or morereagents that tag the compartmentalized protein:cfDNA complexes with acompartment specific molecular barcode comprise reagents for primerextension polymerization (PEP), reagents for ligation or a transposasecomprising a barcoded oligonucleotide.

G8. The method of any one of embodiments G7 to G7.2, further comprisingone or more reagents to affinity purify the protein:cfDNA complexesprior to compartmentalization.

G9. The method of any one of embodiments G1-G4.1, wherein one or morereagents that preserve spatial-proximal contiguity information compriseTn5 bound to a solid phase.

G9.1. The method of embodiment G9, wherein the Tn5 comprises a virtualcompartment specific molecular barcode.

G10. The method of any one of embodiments, G1 to G9.1, wherein step (d)generates cell free DNA with preserved spatial-proximal contiguityinformation and the cell free DNA with preserved spatial-proximalcontiguity information is subjected to bisulfite treatment to generatebisulfite treated cell free DNA with preserved spatial-proximalcontiguity information.

G11. The method of embodiment G10, wherein the bisulfite treated cellfree DNA with preserved spatial-proximal contiguity information issequenced to determine the methylation status of the cell free DNA withpreserved spatial-proximal contiguity information.

G12. The method of any one of embodiments G1 to G9.1, wherein step (d)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

G13. The method of embodiment G12, wherein the sequencing is at a depthof 30× or less.

G14. The method of any one of embodiments G1 to G13, wherein the methodis essentially carried out using automated equipment.

G15. The method of any one of embodiments G1 to G14, wherein after step(d) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

G16. The method of embodiment G15, wherein the temperature is about 55°C.

G17. The method of embodiment G16, wherein the temperature is 55° C.

G18. The method of any one of embodiments G1 to G14, wherein after step(d) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

G19. The method of embodiment G18, wherein after step (d) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

H1 A method for preparing nucleic acids from a formalin-fixedparaffin-embedded (FFPE) sample, that preserves spatial-proximalcontiguity information, comprising:

a) providing a formalin-fixed paraffin-embedded sample;

b) de-waxing the sample to produce a de-waxed sample;

c) rehydrating the de-waxed sample, thereby generating ade-waxed/rehydrated sample;

d) contacting the de-waxed/rehydrated sample sample with lysis buffer,thereby generating a lysed sample;

e) contacting the lysed sample with sodium dodecyl sulfate (SDS) at atemperature of 74° C. for 40 minutes, thereby generating a solubilizedand decompacted sample; and

f) contacting the solubilized and decompacted sample with one or morereagents that generate proximity ligated nucleic acid molecules in situ,thereby preserving spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

H1.1. The method of embodiment H1, wherein the formalin-fixedparaffin-embedded (FFPE) sample is a tissue sample.

H2. The method of embodiment H1 or H1.1, wherein the dewaxed/rehydratedsample is contacted with an extracellular matrix protease prior tocontact with the lysis buffer.

H2.1. The method of embodiment H2, wherein the protease is a collagenaseand/or a dispase.

H2.2. The method of embodiment H2.1, wherein the collagenase is ColI,ColIII or ColIV and the dispase is Dispase I.

H3. The method of any one of embodiments, H1 to H2.2, wherein step (f)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are subjected to bisulfite treatment to generatebisulfite treated nucleic acids with preserved spatial-proximalcontiguity information.

H4. The method of embodiment H3, wherein the bisulfite treated nucleicacids with preserved spatial-proximal contiguity information aresequenced to determine the methylation status of the nucleic acids withpreserved spatial-proximal contiguity information.

H5. The method of any one of embodiments H1 to H2.2, wherein step (f)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

H6. The method of embodiment H5, wherein the sequencing is at a depth of30× or less.

H7. The method of embodiment H5 or H6, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature of 74° C. for 40 minutes.

H8. The method of embodiment H7, wherein % of long-range cis readouts isgreater than 40% of the readouts.

H9. The method of any one of embodiments H1 to H2.2, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 4 years to about 20 years.

H10. The method of any one of embodiments H1 to H2.2, wherein theformalin-fixed paraffin-embedded (FFPE) sample has an archival period ofabout 20 years to about 70 years.

H11. The method of embodiment H9 or H10, wherein step (f) generatesnucleic acids with preserved spatial-proximal contiguity information andthe nucleic acids with preserved spatial-proximal contiguity informationare sequenced to produce sequence readouts.

H12. The method of embodiment H11, wherein the sequencing at a depth of30× or less.

H13. The method of embodiment H11 or H12, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature of 74° C. for 40 minutes.

H14. The method of any one of embodiments H1 to H2.2, wherein thenucleic acid obtained from the formalin-fixed paraffin-embedded (FFPE)sample is less than 200 ng.

H15. The method of embodiment H14, wherein step (f) generates nucleicacids with preserved spatial-proximal contiguity information and thenucleic acids with preserved spatial-proximal contiguity information aresequenced to produce sequence readouts.

H16. The method of embodiment H15, wherein the sequencing at a depth of30× or less.

H17. The method of embodiment H15 or H16, wherein the sequence read-outshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature of 74° C. for 40 minutes.

H18. The method of embodiment H17, wherein % of long-range cis readoutsis greater than 40% of the readouts.

H19. The method of any one of embodiments H1 to H18, wherein the methodis essentially carried out using automated equipment.

H20. The method of any one of embodiments H1 to H19, wherein after step(f) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

H21. The method of embodiment H20, wherein the temperature is about 55°C.

H22. The method of embodiment H21, wherein the temperature is 55° C.

H23. The method of any one of embodiments H1 to H19, wherein after step(f) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

H24. The method of embodiment H23, wherein after step (f) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

I1. A method for preparing nucleic acids from a deeply formalin-fixedsample that preserves spatial-proximal contiguity informationcomprising:

a) providing a deeply formalin-fixed sample;

b) contacting the deeply formalin-fixed sample with a denaturingdetergent at a temperature greater than 65° C., thereby generating asolubilized and decompacted sample; and

c) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

I1.1. The method of embodiment I1, wherein the deeply formalin-fixedsample is a tissue sample.

I1.2. The method of embodiment I1 or I1.1, wherein the deeplyformalin-fixed sample is contacted with an extracellular matrix proteaseprior to contact with lysis buffer.

I1.3. The method of embodiment I1.2, wherein the protease is acollagenase and/or a dispase.

I1.4. The method of embodiment I1.3, wherein the collagenase is ColI,ColIII or ColIV and the dispase is Dispase I.

I1.5. The method of any one of embodiments I1 to I1.4, wherein thedeeply formalin-fixed sample is pulverized.

I2. The method of any one of embodiments I1 to I1.5, wherein contactwith the denaturing detergent is for greater than 10 minutes.

I3. The method of embodiment I2, wherein contact with the denaturingdetergent is 15 to 80 minutes.

I3.1. The method of embodiment I3, wherein contact with the denaturingdetergent is 30 to 50 minutes.

I4. The method of embodiment I3.1, wherein contact with the denaturingdetergent is about 40 minutes.

I4.1. The method of embodiment I4, wherein contact with the denaturingdetergent is 40 minutes.

I5. The method of any one of embodiments I1 to I4.1, wherein thetemperature is greater than 65° C. and less than 80° C.

I6. The method of embodiment I5, wherein the temperature is between 70°C. and 80° C.

I7. The method of embodiment I6, wherein the temperature is about 74° C.

I7.1. The method of embodiment I7, wherein the temperature is 74° C.

I7.2. The method of any one of embodiments I1-I1.5, wherein contact withthe denaturing detergent is for 40 minutes at a temperature of 74° C.

I8. The method of any one of embodiments I1 to I7.2, wherein thedetergent is sodium dodecyl sulfate (SDS).

I9. The method of any one of embodiments I1-I8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

I9.1. The method of embodiment I9, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

I9.2. The method of embodiment I9.1, comprising two restrictionendonucleases.

I10. The method of any one of embodiment I9 to I9.2, wherein theproximity ligated nucleic acid molecules are generated in situ.

I11. The method of any one of embodiments I1-I8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with spatially-proximal nucleicacids of the solubilized and decompacted sample to generatespatially-proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

I12. The method of embodiment I11, wherein the solid phase elements aresolid phase substrates functionalized with a nucleic acid crosslinkingagent.

I13. The method of embodiment I11, wherein the solid phase elements aresolid phase substrates functionalized with an affinity purificationmolecule and the nucleic acid molecules are labeled with an affinitypurification marker.

I13.1. The method of any one of embodiments I11 to I13, wherein thespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements is contacted with one or morereagents for compartmentalization and tagging with a molecular barcode.

I13.2. The method of embodiment I13.1, wherein the one or more reagentsfor compartmentalization comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

I13.3. The method of embodiment I13.1 or I13.2, wherein tagging with amolecular barcode is by primer extension polymerization (PEP) or byligation.

I14. The method of embodiment I11, wherein the solid phase elements aresolid phase substrates functionalized with a transposase comprising abarcoded oligonucleotide that generate spatially-proximal nucleic acidcomprising a ligated barcoded oligonucleotide that are complexed to thesolid phase substrate functionalized with a transposase

I14.1. The method of embodiment I14, wherein the transposase is Tn5.

I15. The method of any one of embodiments I1 to I14.1, wherein thedeeply formalin-fixed sample is provided on a solid surface.

I15.1. The method of embodiment I15.1, wherein the solid surface is apathology slide.

I16. The method of any one of embodiments I1 to I8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and attach compartment-specific molecularbarcodes to the nucleic acids of the solubilized and decompacted sample.

I16.1. The method of embodiment I16, wherein the one or more reagentsthat compartmentalize are a microfluidic compartmentalization devicethat produces microfluidic droplets or microtiter plate wells into whichcomplexes are diluted.

I16.2. The method of embodiment I16 or I16.1, wherein the one or morereagents that tag by attaching compartment-specific molecular barcodescomprise reagents for primer extension polymerization (PEP), reagentsfor ligation or a transposase comprising a barcoded oligonucleotide.

I17. The method of any one of embodiments, I1 to I8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

I18. The method of any one of embodiments, I1 to I17, wherein step (c)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are subjected to bisulfite treatment to generatebisulfite treated nucleic acids with preserved spatial-proximalcontiguity information.

I19. The method of embodiment I18, wherein the bisulfite treated nucleicacids with preserved spatial-proximal contiguity information aresequenced to determine the methylation status of the nucleic acids withpreserved spatial-proximal contiguity information.

I20. The method of any one of embodiments I1 to I17, wherein step (c)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

I21. The method of embodiment I20, wherein the sequencing is at a depthof 30× or less.

I22. The method of embodiment I20 or I21, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

I23. The method of embodiment I22, wherein % of long-range cis readoutsis greater than 40% of the readouts.

I24. The method of any one of embodiments I1 to I17, wherein the deeplyformalin-fixed sample has an archival period of about 4 years to about20 years.

I25. The method of any one of embodiments I1 to I17, wherein the deeplyformalin-fixed sample has an archival period of about 20 years to about70 years.

I26. The method of embodiment I24 or I25, wherein step (c) generatesnucleic acids with preserved spatial-proximal contiguity information andthe nucleic acids with preserved spatial-proximal contiguity informationare sequenced to produce sequence readouts.

I27. The method of embodiment I26, wherein the sequencing at a depth of30× or less.

I28. The method of embodiment I26 or I27, wherein the sequence readoutshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

I29. The method of any one of embodiments I1 to I17, wherein the nucleicacid obtained from the deeply formalin-fixed sample is less than 200 ng.

I30. The method of embodiment I29, wherein step (c) generates nucleicacids with preserved spatial-proximal contiguity information and thenucleic acids with preserved spatial-proximal contiguity information aresequenced to produce sequence readouts.

I31. The method of embodiment I30, wherein the sequencing at a depth of30× or less.

I32. The method of embodiment I30 or I31, wherein the sequence read-outshave a % of long-range cis readouts greater than the % of long-range cisreadouts produced without contact with a denaturing detergent at atemperature greater than 65° C.

I33. The method of embodiment I32, wherein % of long-range cis readoutsis greater than 40% of the readouts.

I34. The method of any one of embodiments I1 to I33, wherein the methodis essentially carried out using automated equipment.

I35. The method of any one of embodiments I1 to I34, wherein after step(c) crosslinking is reversed by contacting the sample with proteinase Kat a temperature of less than 68° C. for about 30 minutes.

I36. The method of embodiment I35, wherein the temperature is about 55°C.

I37. The method of embodiment I36, wherein the temperature is 55° C.

I38. The method of any one of embodiments I1 to I34, wherein after step(c) crosslinking is reversed by incubating the sample at a temperatureof about 95° C. for about 1 hour in the absence of proteinase K.

I39. The method of embodiment I38, wherein after step (c) crosslinkingis reversed by incubating the sample at a temperature of 95° C. for 1hour in the absence of proteinase K.

J1. A kit comprising:

a dewaxing reagent;

a lysis buffer;

a denaturing detergent; and

one or more reagents that preserve spatial-proximal contiguityinformation.

J1.1. The kit of embodiment J1, wherein the dewaxing reagent is xyleneor mineral oil.

J1.2. The kit of embodiment J1 or J1.1, wherein the lysis buffercomprises one or more salts, a protease inhibitor and a non-ionic,non-denaturing detergent.

J1.3. The kit of any one of embodiments J1 to J1.2, wherein thedenaturing detergent is sodium dodecyl sulfate (SDS).

J1.4. The kit of any one of embodiments J1 to J1.3, comprising a reagentto quench the denaturing-detergent.

J1.5. The kit of embodiment J1.4, wherein the reagent is TritonX-100.

J1.6. The kit of embodiment of any one of embodiments J1 to J1.5,comprising an extracellular matrix protease.

J1.7. The kit of embodiment J1.6, wherein the protease is a collagenaseand/or a dispase.

J1.8. The kit of embodiment J1.7, wherein the collagenase is ColI,ColIII or ColIV, and the dispase is Dispase I.

J1.9. The kit of any one of embodiments J1 to J1.8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

J1.9.1. The kit of embodiment J1.9, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

J1.9.2. The kit of embodiment J1.9.1, comprising two restrictionendonucleases.

J1.10. The kit of any one of embodiments J1 to J1.8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements.

J1.10.1. The kit of embodiment J1.10, wherein the solid phase elementsare beads.

J1.11. The kit of embodiment J1.10 or J1.10.1, wherein the solid phaseelements are solid phase substrates functionalized with a nucleic acidcrosslinking reagent.

J1.12. The kit of embodiment J1.11, wherein the crosslinking reagent ispsoralen.

J1.13. The kit of embodiment J1.10 or J1.10.1, wherein the solid phaseelements are solid phase substrates functionalized with an affinitypurification molecule.

J1.13.1. The kit of embodiment J1.13, wherein the affinity purificationmolecule is streptavidin.

J1.13.2. The kit of embodiment J1.13 or J1.13.1, comprising an affinitypurification marker.

J1.13.3. The kit of embodiment J1.13.2, wherein the affinitypurification marker is biotin.

J1.14. The kit of embodiment J1.10 or J1.10.1, wherein the solid phaseelements are solid phase substrates functionalized with a transposasecomprising a barcoded oligonucleotide.

J1.15. The kit of embodiment J1.14, wherein the transposase is Tn5.

J1.16. The kit of any one of embodiments J1 to J1.8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisecompartment-specific molecular barcodes, reagents that tag by attachingcompartment-specific molecular barcodes and/or reagents thatcompartmentalize.

J1.16.1. The kit of embodiment J1.16, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

J1.16.2. The kit of embodiment J1.16 or J1.16.1, wherein the one or morereagents that tag by attaching compartment-specific molecular barcodescomprise reagents for primer extension polymerization (PEP), reagentsfor ligation or a transposase comprising a barcoded oligonucleotide.

J1.17. The kit of any one of embodiments J1 to J1.8, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

J1.17.1. The kit of embodiment J1.17, wherein the Tn5 tetramer comprisesa biotinylated linker sequence.

J1.18. The kit of any one of embodiments J1 to J1.17.1, comprising apathology slide.

J1.19. The kit of any one of embodiments J1 to J1.18, comprising abisulfite reagent.

J2. A kit comprising:

a dewaxing reagent;

a denaturing detergent; and

one or more reagents that preserve spatial-proximal contiguityinformation.

J2.1. The kit of embodiment J2, wherein the dewaxing reagent is xyleneor mineral oil.

J2.2. The kit of embodiment J2 or J2.1, wherein the denaturing detergentis sodium dodecyl sulfate (SDS).

J2.3. The kit of any one of embodiments J2 to J2.2, comprising a reagentto quench the denaturing detergent.

J2.4. The kit of embodiment J2.3, wherein the reagent is TritonX-100.

J2.5. The kit of embodiment of any one of embodiments J2 to J2.4,comprising an extracellular matrix protease.

J2.6. The kit of embodiment J2.5, wherein the protease is a collagenaseand/or a dispase.

J2.7. The kit of embodiment J2.6, wherein the collagenase is ColI,ColIII or ColIV, and the dispase is Dispase I.

J2.8. The kit of any one of embodiments J2 to J2.7, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

J2.8.1. The kit of embodiment J2.8, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

J2.8.2. The kit of embodiment J2.8.1, comprising two restrictionendonucleases.

J2.9. The kit of any one of embodiments J2 to J2.7, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements.

J2.9.1. The kit of embodiment J2.9, wherein the solid phase elements arebeads.

J2.10. The kit of embodiment J2.9 or J2.9.1, wherein the solid phaseelements are solid phase substrates functionalized with a nucleic acidcrosslinking reagent.

J2.11. The kit of embodiment J2.10, wherein the crosslinking reagent ispsoralen.

J2.12. The kit of embodiment J2.9 or J2.9.1, wherein the solid phaseelements are solid phase substrates functionalized with an affinitypurification molecule.

J2.12.1. The kit of embodiment J2.12, wherein the affinity purificationmolecule is streptavidin.

J2.12.2. The kit of embodiment J2.12 or J2.12.1, comprising an affinitypurification marker.

J2.12.3. The kit of embodiment J2.12.2, wherein the affinitypurification marker is biotin.

J2.13. The kit of embodiment J2.9 or J2.9.1, wherein the solid phaseelements are solid phase substrates functionalized with a transposasecomprising a barcoded oligonucleotide.

J2.14. The kit of embodiment J2.13, wherein the transposase is Tn5.

J2.15. The kit of any one of embodiments J2 to J2.7, wherein one or morereagents that preserve spatial-proximal contiguity information comprisecompartment-specific molecular barcodes, reagents that tag by attachingcompartment-specific molecular barcodes and/or reagents thatcompartmentalize.

J2.15.1. The kit of embodiment J2.15, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

J2.15.2. The kit of embodiment J2.15. or J2.15.1, wherein the one ormore reagents that tag by attaching compartment-specific molecularbarcodes comprise reagents for primer extension polymerization (PEP),reagents for ligation or a transposase comprising a barcodedoligonucleotide.

J2.16. The kit of any one of embodiments J2 to J2.7, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

J2.16.1. The kit of embodiment J2.16, wherein the Tn5 tetramer comprisesa biotinylated linker sequence.

J2.17. The kit of any one of embodiments J2 to J2.16.1, comprising apathology slide.

J2.18. The kit of any one of embodiments J2 to J2.17, comprising abisulfite reagent.

J3. A kit comprising:

a lysis buffer;

a denaturing detergent; and

one or more reagents that preserve spatial-proximal contiguityinformation.

J3.1. The kit of embodiment J3, wherein the lysis buffer comprises oneor more salts, a protease inhibitor and a non-ionic, non-denaturingdetergent.

J3.2. The kit of embodiment J3 or J3.1, comprising a reagent to quenchthe denaturing detergent.

J3.3. The kit of embodiment J3.2, wherein the reagent is TritonX-100.

J3.4. The kit of any one of embodiments J3 to J3.3, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

J3.5. The kit of embodiment J3.4, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

J3.5.1. The kit of embodiment J3.5, comprising two restrictionendonucleases.

J3.6. The kit of any one of embodiments J3 to J3.3, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements.

J3.6.1. The kit of embodiment J3.6, wherein the solid phase elements arebeads.

J3.7. The kit of embodiment J3.6 or J3.6.1, wherein the solid phaseelements are solid phase substrates functionalized with a nucleic acidcrosslinking reagent.

J3.8. The kit of embodiment J3.7, wherein the crosslinking reagent ispsoralen.

J3.9. The kit of embodiment J3.6 or J3.6.1, wherein the solid phaseelements are solid phase substrates functionalized with an affinitypurification molecule.

J3.9.1. The kit of embodiment J3.9, wherein the affinity purificationmolecule is streptavidin.

J3.9.2. The kit of embodiment J3.9 or J3.9.1, comprising an affinitypurification marker.

J3.9.3. The kit of embodiment J3.9.2, wherein the affinity purificationmarker is biotin.

J3.10. The kit of embodiment J3.6 or J3.6.1, wherein the solid phaseelements are solid phase substrates functionalized with a transposasecomprising a molecular barcoded oligonucleotide.

J3.11. The kit of embodiment J3.10, wherein the transposase is Tn5.

J3.12. The kit of any one of embodiments J3 to J3.3, wherein one or morereagents that preserve spatial-proximal contiguity information comprisecompartment-specific molecular barcodes, reagents that tag by attachingcompartment-specific molecular barcodes and/or reagents thatcompartmentalize.

J3.12.1. The kit of embodiment J3.12, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

J3.12.2. The kit of embodiment J3.12 or J3.12.1, wherein the one or morereagents that tag by attaching compartment-specific molecular barcodescomprise reagents for primer extension polymerization (PEP), reagentsfor ligation or a transposase comprising a barcoded oligonucleotide.

J3.13. The kit of any one of embodiments J3 to J3.3, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

J3.13.1. The kit of embodiment J3.13, wherein the Tn5 tetramer comprisesa biotinylated linker sequence.

J3.14. The kit of any one of embodiments J3 to J3.13.1, comprising apathology slide.

J3.15. The kit of any one of embodiments J3 to J3.14, comprising abisulfite reagent.

J3.16. The kit of embodiment of any one of embodiments J3 to J3.15,comprising an extracellular matrix protease.

J3.17. The kit of embodiment J3.16, wherein the protease is acollagenase and/or a dispase.

J3.18. The kit of embodiment J3.17, wherein the collagenase is ColI,ColIII or ColIV, and the dispase is Dispase I.

J4. A kit comprising:

a denaturing detergent; and

one or more reagents that preserve spatial-proximal contiguityinformation.

J4.1. The kit of embodiment J4, wherein the denaturing detergent issodium dodeccyl sulfate (SDS).

J4.2. The kit of embodiment J4 or J4.1, comprising a reagent to quenchthe denaturing detergent.

J4.3. The kit of embodiment J4.2, wherein the reagent is TritonX-100.

J4.4. The kit of any one of embodiments J4 to J4.3, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

J4.5. The kit of embodiment J4.4, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

J4.5.1. The kit of embodiment J4.5, comprising two restrictionendonucleases.

J4.6. The kit of any one of embodiments J4 to J4.3, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements.

J4.7. The kit of embodiment J4.6, wherein the solid phase elements arebeads.

J4.8. The kit of embodiment J4.6 or J4.7, wherein the solid phaseelements are solid phase substrates functionalized with a nucleic acidcrosslinking reagent.

J4.9. The kit of embodiment J4.8, wherein the crosslinking reagent ispsoralen.

J4.10. The kit of embodiment J4.6 or J4.7, wherein the solid phaseelements are solid phase substrates functionalized with an affinitypurification molecule.

J4.10.1. The kit of embodiment J4.10, wherein the affinity purificationmolecule is streptavidin

J4.10.2. The kit of embodiment J4.10 or J4.10.1, comprising an affinitypurification marker.

J4.10.3. The kit of embodiment J4.10.2, wherein the affinitypurification marker is biotin.

J4.11. The kit of embodiment J4.6 or J4.7, wherein the solid phaseelements are solid phase substrates functionalized with a transposasecomprising a barcoded oligonucleotide.

J4.12. The kit of embodiment J4.11, wherein the transposase is Tn5.

J4.13. The kit of any one of embodiments J4 to J4.3, wherein one or morereagents that preserve spatial-proximal contiguity information comprisecompartment-specific molecular barcodes, reagents that tag by attachingcompartment-specific molecular barcodes and/or reagents thatcompartmentalize.

J4.13.1. The kit of embodiment J4.13, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

J4.13.2. The method of embodiment J4.13 or J4.13.1, wherein the one ormore reagents that tag by attaching compartment-specific molecularbarcodes comprise reagents for primer extension polymerization (PEP),reagents for ligation or a transposase comprising a barcodedoligonucleotide.

J4.13.3. The kit of any one of embodiments J4.13 to J4.13.2, furthercomprising one or more reagents that affinity purify native spatiallyproximal nucleic acid molecules.

J4.14. The kit of any one of embodiments J4 to J4.3, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

J4.14.1. The kit of embodiment J4.14, wherein the Tn5 tetramer comprisesa biotinylated linker sequence.

J4.15. The kit of any one of embodiments J4 to J4.14.1, comprising apathology slide.

J4.16. The kit of any one of embodiments J4 to J4.15, comprising abisulfite reagent.

J4.17. The kit of embodiment of any one of embodiments J4 to J4.16,comprising an extracellular matrix protease.

J4.18. The kit of embodiment J4.17, wherein the protease is acollagenase and/or a dispase.

J4.19. The kit of embodiment J4.18, wherein the collagenase is ColI,ColIII or ColIV, and the dispase is Dispase I.

J5. A kit comprising:

a solid phase; and

one or more reagents that preserve spatial-proximal contiguityinformation.

J5.1. The kit of embodiment J5, wherein the solid phase comprises acarboxylated surface.

J5.2. The kit of embodiment J5.1, wherein the solid phase comprises amicroplate or a bead.

J5.3. The kit of embodiment J5, wherein the solid phase is coated with across-linking reagent.

J5.4. The kit of embodiment J5.3, wherein the cross-linking reagent ispsoralen.

J5.5. The kit of embodiment J5.3 or 15.4, wherein the solid phase is amagnetic bead.

J5.6. The kit of any one of embodiments J5 to J5.2, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

J5.7. The kit of embodiment J5.6, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: a restriction endonuclease, a DNA polymerase, aplurality of nucleotides comprising at least one biotinylatednucleotide, and a ligase.

J5.8. The kit of any one of embodiments J5 to J5.5, wherein the one ormore reagents that preserve spatial-proximal contiguity informationcomprise a Tn5 tetramer.

J5.9. The kit of embodiment J5.8, wherein the Tn5 tetramer comprises abiotinylated linker sequence.

J5.10. The kit of any one of embodiments J5 to J5.4 wherein one or morereagents that preserve spatial-proximal contiguity information comprisecompartment-specific molecular barcodes, reagents that tag by attachingcompartment-specific molecular barcodes and/or reagents thatcompartmentalize.

J5.11. The kit of embodiment J5.10, wherein the one or more reagentsthat compartmentalize comprise a microfluidic compartmentalizationdevice that produces microfluidic droplets or microtiter plate wellsinto which complexes are diluted.

J5.11.1. The kit of embodiment J5.10 or J5.11, wherein the one or morereagents that tag with a compartment specific molecular barcode comprisereagents for primer extension polymerization (PEP), reagents forligation or a transposase comprising a barcoded oligonucleotide.

J5.11.2. The kit of any one of embodiments J5.10 to J5.11.1, furthercomprising one or more reagents that affinity purify native spatiallyproximal nucleic acid molecules.

J5.12. The kit of any one of embodiments J5 to J5.2, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea transposome comprising a compartment-specific barcoded oligonucleotideand the transposome comprising a compartment-specific barcodedoligonucleotide is linked to a solid phase element.

J5.13. The kit of embodiment J15.12, wherein the transposome is Tn5.

J5.14. The kit of any one of embodiments J5 to J5.13, comprising abisulfite reagent.

K1. A method for reversing crosslinking in a sample crosslinked topreserve spatial-proximal contiguity information, wherein crosslinkingis reversed by contacting the sample with proteinase K at a temperatureof less than 68° C. for about 30 minutes.

K2. The method of embodiment K1, wherein the temperature is about 55° C.

K3. The method of embodiment K2, wherein the temperature is 55° C.

L1. A method for reversing crosslinking in a sample crosslinked topreserve spatial-proximal contiguity information, wherein crosslinkingis reversed by incubating the sample at a temperature of about 95° C.for about 1 hour in the absence of proteinase K.

L2. The method of embodiment L1, wherein crosslinking is reversed byincubating the sample at a temperature of 95° C. for 1hour in theabsence of proteinase K.

M1. A method for preparing nucleic acids from a formalin-fixedparaffin-embedded (FFPE) sample of cells, that preservesspatial-proximal contiguity information, comprising:

a) providing a formalin-fixed paraffin-embedded sample of cells;

b) de-waxing the sample to produce a de-waxed sample;

c) rehydrating the de-waxed sample, thereby generating ade-waxed/rehydrated sample;

d) contacting the de-waxed/rehydrated sample with lysis buffer; therebygenerating a lysed sample;

e) contacting the lysed sample; with a denaturing detergent at atemperature of about 62° C. for greater than 10 minutes, therebygenerating a solubilized and decompacted sample; and

f) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

M1.1. The method of embodiment M1, wherein the temperature is 62° C.

M2. The method of embodiment M1 or M1.1, wherein contact with thedenaturing detergent is 15 to 80 minutes.

M3. The method of embodiment M2, wherein contact with the denaturingdetergent is 30 to 50 minutes

M4. The method of embodiment M3, wherein contact with the denaturingdetergent about 40 minutes.

M5. The method of embodiment M4, wherein contact with the denaturingdetergent is 40 minutes.

M6. The method of any one of embodiments M1 to M5, wherein the detergentis sodium dodecyl sulfate (SDS).

M7. The method of any one of embodiments M1 to M6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

M8. The method of embodiment M7, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

M9. The method of embodiment M8, comprising two restrictionendonucleases.

M10. The method of any one of embodiments M7 to M9, wherein theproximity ligated nucleic acid molecules are generated in situ.

M11. The method of any one of embodiments M1 to M6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with the spatially proximalnucleic acid of the solubilized and decompacted sample to generatespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

M12. The method of any one of embodiments M1 to M6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and tag by attaching compartment-specificmolecular barcodes to the nucleic acids of the solubilized anddecompacted sample.

M13. The method of any one of embodiments M1 to M6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

M14. The method of any one of embodiments M1 to M13, wherein step (f)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

M15. The method of any one of embodiments M1 to M14, wherein thedewaxing/rehydration of the sample is omitted.

N1. A method for preparing nucleic acids from a formalin-fixedparaffin-embedded (FFPE) sample of cells, that preservesspatial-proximal contiguity information, comprising:

a) providing a formalin-fixed paraffin-embedded sample of cells;

b) de-waxing the sample to produce a de-waxed sample;

c) rehydrating the de-waxed sample, thereby generating ade-waxed/rehydrated sample;

d) contacting the de-waxed/rehydrated sample with a denaturing detergentat a temperature of about 62° C. for greater than 10 minutes, therebygenerating a solubilized and decompacted sample; and

e) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

N1.1. The method of embodiment N1, wherein the temperature is 62° C.

N2. The method of embodiment N1 or N1.1, wherein contact with thedenaturing detergent is 15 to 80 minutes.

N3. The method of embodiment N2, wherein contact with the denaturingdetergent is 30 to 50 minutes

N4. The method of embodiment N3, wherein contact with the denaturingdetergent about 40 minutes.

N5. The method of embodiment N4, wherein contact with the denaturingdetergent is 40 minutes.

N6. The method of any one of embodiments N1 to N5, wherein the detergentis sodium dodecyl sulfate (SDS).

N7. The method of any one of embodiments N1 to N6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

N8. The method of embodiment N7, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

N9. The method of embodiment N8, comprising two restrictionendonucleases.

N10. The method of any one of embodiments N7 to N9, wherein theproximity ligated nucleic acid molecules are generated in situ.

N11. The method of any one of embodiments N1 to N6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with the spatially proximalnucleic acid of the solubilized and decompacted sample to generatespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

N12. The method of any one of embodiments N1 to N6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and tag by attaching compartment-specificmolecular barcodes to the nucleic acids of the solubilized anddecompacted sample.

N13. The method of any one of embodiments N1 to N6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

N14. The method of any one of embodiments N1 to N13, wherein step (e)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

N15. The method of any one of embodiments N1 to N14, wherein thedewaxing/rehydration of the sample is omitted.

O1. A method for preparing nucleic acids from a deeply formalin-fixedsample of cells that preserves spatial-proximal contiguity informationcomprising:

a) providing a deeply formalin-fixed sample of cells;

b) contacting the deeply formalin-fixed sample with lysis buffer,thereby generated a lysed sample;

c) contacting the lysed sample with a denaturing detergent at atemperature of about 62° C. for greater than 10 minutes, therebygenerating a solubilized and decompacted sample; and

d) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

O1.1. The method of embodiment O1, wherein the temperature is 62° C.

O2. The method of embodiment O1 or O1.1, wherein contact with thedenaturing detergent is 15 to 80 minutes.

O3. The method of embodiment O2, wherein contact with the denaturingdetergent is 30 to 50 minutes

O4. The method of embodiment O3, wherein contact with the denaturingdetergent about 40 minutes.

O5. The method of embodiment O4, wherein contact with the denaturingdetergent is 40 minutes.

O6. The method of any one of embodiments O1 to O5, wherein the detergentis sodium dodecyl sulfate (SDS).

O7. The method of any one of embodiments O1 to O6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

O8. The method of embodiment O7, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

O9. The method of embodiment O8, comprising two restrictionendonucleases.

O10. The method of any one of embodiments O7 to O9, wherein theproximity ligated nucleic acid molecules are generated in situ.

O11. The method of any one of embodiments O1 to O6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with the spatially proximalnucleic acid of the solubilized and decompacted sample to generatespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

O12. The method of any one of embodiments O1 to O6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and tag by attaching compartment-specificmolecular barcodes to the nucleic acids of the solubilized anddecompacted sample.

O13. The method of any one of embodiments O1 to O6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

O14. The method of any one of embodiments O1 to O13, wherein step (d)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

P1. A method for preparing nucleic acids from a deeply formalin-fixedsample of cells that preserves spatial-proximal contiguity informationcomprising:

a) providing a deeply formalin-fixed sample of cells;

b) contacting the deeply formalin-fixed sample with a denaturingdetergent at a temperature of about 62° C. for greater than 10 minutes,thereby generating a solubilized and decompacted sample; and

c) contacting the solubilized and decompacted sample with one or morereagents that preserve spatial-proximal contiguity information in thenucleic acids of the solubilized and decompacted sample.

P1.1. The method of embodiment P1, wherein the temperature is 62° C.

P2. The method of embodiment P1 or P1.1, wherein contact with thedenaturing detergent is 15 to 80 minutes.

P3. The method of embodiment P2, wherein contact with the denaturingdetergent is 30 to 50 minutes

P4. The method of embodiment P3, wherein contact with the denaturingdetergent about 40 minutes.

P5. The method of embodiment P4, wherein contact with the denaturingdetergent is 40 minutes.

P6. The method of any one of embodiments P1 to P5, wherein the detergentis sodium dodecyl sulfate (SDS).

P7. The method of any one of embodiments P1 to P6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that generate proximity ligated nucleic acid molecules.

P8. The method of embodiment P7, wherein the reagents that generateproximity ligated nucleic acid molecules comprise one or more of thefollowing reagents: at least one restriction endonuclease, a DNApolymerase, a plurality of nucleotides comprising at least onebiotinylated nucleotide, and a ligase.

P9. The method of embodiment P8, comprising two restrictionendonucleases.

P10. The method of any one of embodiments P7 to P9, wherein theproximity ligated nucleic acid molecules are generated in situ.

P11. The method of any one of embodiments P1 to P6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisesolid phase elements that form complexes with the spatially proximalnucleic acid of the solubilized and decompacted sample to generatespatially proximal nucleic acid of the solubilized and decompactedsample complexed to solid phase elements.

P12. The method of any one of embodiments P1 to P6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisereagents that compartmentalize and tag by attaching compartment-specificmolecular barcodes to the nucleic acids of the solubilized anddecompacted sample.

P13. The method of any one of embodiments P1 to P6, wherein one or morereagents that preserve spatial-proximal contiguity information comprisea Tn5 tetramer.

P14. The method of any one of embodiments P1 to P13, wherein step (c)generates nucleic acids with preserved spatial-proximal contiguityinformation and the nucleic acids with preserved spatial-proximalcontiguity information are sequenced to produce sequence readouts.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents. Their citation is not an indication of asearch for relevant disclosures. All statements regarding the date(s) orcontents of the documents is based on available information and is notan admission as to their accuracy or correctness.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent technology has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

What is claimed:
 1. A method for detecting the presence or absence of achromosome rearrangement in a sample, the method comprising: a)selecting a sample, wherein one or more genes associated with cancer inthe sample were analyzed for one or more genomic variants associatedwith cancer, and the one or more genes associated with cancer compriseno detectable genomic variant associated with cancer; b) performing anucleic acid analysis on the selected sample, wherein the analysiscomprises a method that preserves spatial-proximal contiguityinformation; and c) detecting whether a chromosome rearrangement ispresent or absent in the selected sample according to the nucleic acidanalysis in (b).
 2. The method of claim 1, wherein a breakpoint of thechromosome rearrangement is within the one or more genes analyzed in(a).
 3. The method of claim 1, wherein the one or more genes wereanalyzed for the one or more genomic variants according to genome-widesequencing and/or targeted sequencing.
 4. The method of claim 3, whereinthe targeted sequencing comprises one or more of RNA-Seq, oncology genepanel sequencing, and capture sequencing
 5. The method of claim 1,wherein the one or more genomic variants comprise mutations and/ortranslocations.
 6. The method of claim 1, wherein the chromosomerearrangement comprises a translocation.
 7. The method of claim 6,wherein the translocation is an intra-chromosome translocation.
 8. Themethod of claim 6, wherein the translocation is an inter-chromosometranslocation.
 9. The method of claim 1, wherein the nucleic acidanalysis in (b) comprises generating proximity ligated nucleic acidmolecules.
 10. The method of claim 9, wherein the nucleic acid analysisin (b) further comprises sequencing the proximity ligated nucleic acidmolecules.
 11. The method of claim 10, wherein the sequencing isperformed at 30×, 15×, 5×, or 1×.
 12. The method of claim 10, whereinthe sequencing is performed at up to about 10×.
 13. The method of claim10, wherein the sequencing is performed at 0.75×, 0.25×, or 0.05×. 14.The method of claim 1, wherein the sample is a tissue sample, a cellsample, a blood sample, or a urine sample.
 15. The method of claim 1,wherein the sample comprises FFPE tissue, frozen cells, or cell-freenucleic acid.
 16. The method of claim 1, wherein the sample comprisestumor tissue.
 17. The method of claim 1, wherein the sample comprisesFFPE tumor tissue.
 18. The method of claim 16, wherein the tumor is agastrointestinal stromal tumor (GIST) or a posterior fossa ependymoma(PFE) tumor.
 19. The method of claim 1, further comprising extractingDNA from the sample.
 20. The method of claim 19, wherein <200 ng of DNAis extracted from the sample.